CN110317824B - Preparation method of blood pressure-reducing rice material, and gene and protein used in preparation method - Google Patents

Preparation method of blood pressure-reducing rice material, and gene and protein used in preparation method Download PDF

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CN110317824B
CN110317824B CN201810270131.XA CN201810270131A CN110317824B CN 110317824 B CN110317824 B CN 110317824B CN 201810270131 A CN201810270131 A CN 201810270131A CN 110317824 B CN110317824 B CN 110317824B
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曲乐庆
钱丹丹
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Institute of Botany of CAS
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Abstract

The invention discloses a preparation method of a blood pressure-reducing rice material, and a gene and a protein used in the preparation method. The invention discloses a preparation method of a blood pressure reducing rice material, which comprises the following steps: improving the expression level of the protein related to blood pressure reduction in the target plant to obtain the plant with the function of reducing the animal blood pressure; the blood pressure lowering related protein is obtained by directly connecting n angiotensin converting enzyme inhibitory peptides or connecting the n angiotensin converting enzyme inhibitory peptides with connecting peptides; n is a natural number, and n is more than or equal to 1; the blood pressure lowering related protein contains m angiotensin converting enzyme inhibitory peptides; m is a natural number, and m is more than or equal to 2; the protein related to the blood pressure reduction contains a recognition sequence of protease between every two angiotensin converting enzyme inhibitory peptides. Experiments prove that the blood pressure reducing rice material can effectively reduce the blood pressure of hypertensive animals.

Description

Preparation method of blood pressure-reducing rice material, and gene and protein used in preparation method
Technical Field
The invention relates to a preparation method of a rice material for reducing blood pressure, a gene and a protein used in the preparation method, belonging to the field of plant genetic engineering.
Background
Hypertension is a common cardiovascular disease, which can cause damage to the brain, kidney and cardiovascular system, and is an important factor causing diseases such as cerebral apoplexy, coronary heart disease and heart failure. Angiotensin Converting Enzyme (ACE) plays a key role in the process of blood pressure regulation, and it converts inactive Angiotensin I to active Angiotensin II. Angiotensin II is the strongest vasoconstrictor in the body and also stimulates aldosterone secretion, directly causing blood pressure rise. And simultaneously, the angiotensin converting enzyme can also degrade bradykinin with the vasodilatation effect, so that the blood pressure is indirectly increased. Angiotensin converting enzyme is therefore a primary target for the prevention and treatment of hypertension.
Hypertension is currently treated mainly by drug therapy, and hypotensive drugs are mainly angiotensin converting enzyme inhibitors. Since the first synthetic orally available peptide ACE inhibitor Captopril (Captopril) appeared in 1981, a number of chemically synthesized ACE inhibitors have been used clinically. However, the antihypertensive drugs have short action time and long treatment period, can cause blood pressure rebound after drug withdrawal, have high cost and great side effect, and can cause renal function damage, hypotension and the like. Therefore, a novel antihypertensive drug which is safe, long-acting and free from toxic and side effects is urgently needed to be searched.
Research in recent years shows that some short peptides in food protein enzymatic hydrolysate have the function of reducing blood pressure, such as rice, corn, barley, soybean, sesame and other crop seeds, and casein, pig hemoglobin, whey protein, yoghourt, fish protein, cheese, pork, chicken protein and other Angiotensin Converting Enzyme Inhibitory Peptides (ACEIP) which are composed of 2-12 amino acids and have the function of reducing blood pressure. The antihypertensive active peptides have high edible safety and no toxic or side effect, and have the common outstanding advantages of playing a role of reducing blood pressure for patients with hypertension and having no function of reducing blood pressure for people with normal blood pressure; besides the function of reducing blood pressure, the health-care food also has the functions of resisting oxidation, promoting digestion, reducing blood sugar, resisting platelet aggregation, enhancing human immunity and the like; has the advantages of high thermal stability, water solubility and the like, thereby being capable of being used as a functional factor to be added into beverages and foods and having good market prospect.
At present, research finds that many ACE inhibitory peptides derived from food, such as DKIHPF, YQQPVL, FFVAPFPEVFGK, IPP, YLAKKAPHMHIR, VPP, SKVYPFPGPI, LKPMM, KVLPVPE and the like, are all derived from milk protein and have good ACE inhibitory activity.
Although dietary proteins widely contain ACEIP having an effect of inhibiting blood pressure elevation, these ACEIPs form active peptides having a blood pressure lowering effect through a complicated protease hydrolysis process, and thus even if a certain protein contains ACEIP components, it is often impossible to form active peptides having physiological functions through appropriate protease hydrolysis to exert the blood pressure lowering function. Meanwhile, the ACEIP content in the natural protein is very low, and the ACEIP production by utilizing the natural protein through protease hydrolysis and recovery has high cost, so that the industrialization can not be realized.
Disclosure of Invention
The invention aims to solve the technical problem of how to prepare the plant material with the function of reducing the blood pressure of animals.
In order to solve the above technical problems, the present invention provides a method for preparing a plant having a function of lowering blood pressure of an animal, the method comprising: improving the expression level of the protein related to blood pressure reduction in the target plant to obtain the plant with the function of reducing the animal blood pressure;
the protein related to the blood pressure reduction is obtained by directly connecting n angiotensin converting enzyme inhibitory peptides or connecting the peptides; n is a natural number, and n is more than or equal to 1;
the blood pressure lowering related protein contains m angiotensin converting enzyme inhibitory peptides; m is a natural number, and m is more than or equal to 2;
and a recognition sequence of protease is contained between every two angiotensin converting enzyme inhibitory peptides of the blood pressure lowering related protein.
The Angiotensin Converting Enzyme Inhibitory Peptide (ACEIP) is a short peptide with blood pressure lowering function in food protein enzymolysis products, such as polypeptide with blood pressure lowering function composed of 2-12 amino acids derived from crop seeds of rice, corn, barley, soybean, sesame and the like, casein, pig hemoglobin, whey protein, yoghourt, fish protein, cheese, pork, chicken protein and the like.
The recognition sequence of the protease may be formed at the junction by directly linking two angiotensin-converting enzyme-inhibiting peptides, or may be formed at the junction by linking two angiotensin-converting enzyme-inhibiting peptides via the linking peptide.
The protein related to blood pressure reduction can be subjected to enzyme digestion by the protease to obtain q polypeptides; q is less than or equal to m; the polypeptide is a1) or a2) as follows:
a1) the angiotensin-converting enzyme inhibitory peptide;
a2) and a polypeptide obtained by adding 1-2 amino acid residues to the N-terminal and/or C-terminal of the angiotensin-converting enzyme inhibitory peptide.
The protease may be the animal protease.
In the above method, the protease may be trypsin or pepsin.
The linker peptide may be Gln-Arg.
n can satisfy: n is more than or equal to 1 and less than or equal to 20. Further, n may satisfy: n is more than or equal to 1 and less than or equal to 10.
m can satisfy: m is more than or equal to 2 and less than or equal to 20.
In the above method, the angiotensin-converting enzyme inhibitory peptide may be at least one of the following polypeptides a1) -a10), but is not limited to the polypeptides a1) -a 10):
A1) a polypeptide shown in positions 3-8 of sequence 1;
A2) a polypeptide shown in 11 th to 16 th positions of the sequence 1;
A3) a polypeptide shown in 19 th to 30 th positions of the sequence 1;
A4) a polypeptide shown in 31 st to 33 rd positions of the sequence 1;
A5) a polypeptide shown in positions 34-40 of sequence 1;
A6) a polypeptide shown in positions 43-47 of sequence 1;
A7) a polypeptide shown in positions 50-61 of sequence 1;
A8) a polypeptide shown in positions 64-66 of sequence 1;
A9) a polypeptide as shown in positions 68-73 of sequence 1;
A10) a polypeptide shown in positions 76-85 of sequence 1.
The protein related to the blood pressure reduction can be obtained by directly connecting ten polypeptides of the A1) -A10) or connecting the polypeptides.
In the above method, the protein associated with blood pressure reduction may be B1), B2) or B3) as follows:
B1) the amino acid sequence is the protein of sequence 1;
B2) the protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequence shown in the sequence 1 in the sequence table and has the same function;
B3) a fusion protein obtained by connecting a label to the N terminal or/and the C terminal of B1) or B2).
In order to facilitate the purification of the protein in B1), the amino terminal or the carboxyl terminal of the protein consisting of the amino acid sequence shown in the sequence 1 in the sequence table can be connected with the tags shown in the table 1.
TABLE 1 sequence of tags
Label (R) Residue of Sequence of
Poly-Arg 5-6 (typically 5) RRRRR
Poly-His 2-10 (generally 6) HHHHHH
FLAG 8 DYKDDDDK
Strep-tag II 8 WSHPQFEK
c-myc 10 EQKLISEEDL
The protein according to A2), wherein the substitution and/or deletion and/or addition of one or more amino acid residues is a substitution and/or deletion and/or addition of not more than 10 amino acid residues.
The protein of A2) above may be artificially synthesized, or may be obtained by synthesizing the coding gene and then performing biological expression.
The gene encoding the protein in A2) above can be obtained by deleting one or several amino acid residues from the DNA sequence shown in positions 126-380 of the sequence 2, and/or by carrying out missense mutation of one or several base pairs, and/or by connecting the coding sequence of the tag shown in Table 1 above to the 5 'end and/or 3' end thereof. Wherein, the DNA molecule shown in the 126-380 th position of the sequence 2 encodes the protein shown in the sequence 1.
B3) The tag of (1) may also be a signal peptide. The signal peptide can transport the protein related to the blood pressure reduction to a specific position of a cell. The signal peptide may be a GluA2 signal peptide. The signal peptide can be specifically a polypeptide shown in 1 st to 35 th positions of a sequence 3 in a sequence table.
In the above method, the increase in the expression level of the blood pressure-lowering related protein in the target plant can be achieved by introducing an expression cassette containing a gene encoding the blood pressure-lowering related protein into the target plant.
In the above method, the encoding gene may be c1) or c2) or c3) as follows:
c1) the coding sequence is cDNA molecule or DNA molecule at position 106-360 of sequence 2 in the sequence table;
c2) a cDNA molecule or DNA molecule which has 75 percent or more than 75 percent of identity with the nucleotide sequence defined by c1) and codes the protein related to the blood pressure reduction;
c3) a cDNA molecule or a DNA molecule which is hybridized with the nucleotide sequence limited by the c1) under strict conditions and codes the protein related to the blood pressure reduction.
The nucleotide sequence encoding the protein associated with blood pressure reduction of the present invention can be easily mutated by a person of ordinary skill in the art using known methods, such as directed evolution and point mutation. Those nucleotides which are artificially modified and have 75% or more identity to the nucleotide sequence of the blood pressure lowering-related protein isolated according to the present invention are derived from the nucleotide sequence of the present invention and are identical to the sequence of the present invention as long as they encode the blood pressure lowering-related protein and have the function of the blood pressure lowering-related protein.
The term "identity" as used herein refers to sequence similarity to a native nucleic acid sequence. "identity" includes nucleotide sequences that are 75% or more, or 85% or more, or 90% or more, or 95% or more identical to the nucleotide sequence of a protein consisting of the amino acid sequence shown in coding sequence 1 of the present invention. Identity can be assessed visually or by computer software. Using computer software, the identity between two or more sequences can be expressed in percent (%), which can be used to assess the identity between related sequences.
The stringent conditions are hybridization and washing of the membrane 2 times, 5min each, at 68 ℃ in a solution of 2 XSSC, 0.1% SDS, and 2 times, 15min each, at 68 ℃ in a solution of 0.5 XSSC, 0.1% SDS; alternatively, hybridization was carried out at 65 ℃ in a solution of 0.1 XSSPE (or 0.1 XSSC), 0.1% SDS, and the membrane was washed.
The above-mentioned identity of 75% or more may be 80%, 85%, 90% or 95% or more.
B2) The expression cassette refers to DNA capable of expressing the blood pressure lowering related protein in a host cell, and the DNA not only can comprise a promoter for starting the transcription of the gene coding for the blood pressure lowering related protein, but also can comprise a terminator for stopping the transcription of the gene coding for the blood pressure lowering related protein. Further, the expression cassette may also include an enhancer sequence.
In the above method, the expression of the coding gene in the expression cassette can be initiated by a tissue-specific promoter capable of initiating expression of the coding gene in the edible part of the animal.
The edible part of the animal is the seed of the target plant.
The tissue specific promoter is a GluC promoter. The GluC promoter can start the expression of a target gene in endosperm.
The GluC promoter may be derived from the plant of interest. The GluC promoter may in particular be d1) or d2) or d3) as follows:
d1) DNA molecule of sequence 4 in the sequence table;
d2) a DNA molecule which has 75 percent or more than 75 percent of identity with the nucleotide sequence defined by d1) and has the same function;
d3) a DNA molecule which is hybridized with the nucleotide sequence limited by d1) under strict conditions and has the same function.
The expression cassette also contains a terminator. The terminator may be derived from the plant of interest. The terminator may specifically be a GluB5 terminator. The GluB5 terminator can be e1) or e2) or e3) as follows:
e1) DNA molecule of sequence 5 in the sequence table;
e2) a DNA molecule which has 75 percent or more than 75 percent of identity with the nucleotide sequence defined by e1) and has the same function;
e3) a DNA molecule which is hybridized with the nucleotide sequence limited by the e1) under strict conditions and has the same function.
In practical application, at least two of the blood pressure reduction-related proteins can be connected in series to increase the expression level of the blood pressure reduction-related proteins. In one embodiment of the invention, the expression cassette comprises two of the encoding genes in tandem. The expression cassette further comprises a gene encoding the signal peptide upstream of the two genes encoding the signal peptide connected in series.
In the above method, the plant of interest may be f1) or f2) or f 3):
f1) a monocot or dicot;
f2) a gramineous plant;
f3) rice, maize, wheat, sorghum, oats, barley or rye.
The dicot can be an endosperm dicot.
In order to solve the technical problem, the invention also provides any one of the following products:
G1) the blood pressure lowering related protein;
G2) a biomaterial related to the blood pressure lowering-related protein, the biomaterial being any one of the following G21) to G27):
G21) a nucleic acid molecule encoding the blood pressure lowering related protein;
G22) an expression cassette comprising the nucleic acid molecule of G21);
G23) a recombinant vector containing the nucleic acid molecule of G21) or a recombinant vector containing the expression cassette of G22);
G24) a recombinant microorganism containing G21) the nucleic acid molecule, or a recombinant microorganism containing G22) the expression cassette, or a recombinant microorganism containing G23) the recombinant vector;
G25) a transgenic plant cell line comprising the nucleic acid molecule of G21) or a transgenic plant cell line comprising the expression cassette of G22);
G26) transgenic plant tissue comprising the nucleic acid molecule of G21) or transgenic plant tissue comprising the expression cassette of G22);
G27) a transgenic plant organ containing the nucleic acid molecule of G21), or a transgenic plant organ containing the expression cassette of G22).
Wherein the nucleic acid molecule may be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA, etc.
G21) The nucleic acid molecule may be a gene encoding a gene as described in the above methods.
G22) The expression cassette may be as described in the above methods.
The existing vector can be used for constructing a recombinant vector containing the nucleic acid molecule expression cassette for encoding the protein related to the blood pressure reduction.
The vector may be a plasmid, cosmid, phage or viral vector. The plasmid may be specifically pGPTV-pGluc-tGluB5 expression vector.
G23) The recombinant vector can be pGPTV-SPAA; the pGPTV-SPAA is a recombinant vector obtained by replacing a DNA fragment between SalI and SacI recognition sequences of a pGPTV-pGluc-tGluB5 expression vector with a DNA fragment shown in a sequence 2, and the recombinant vector can express two fusion proteins of SPAA and the signal peptide which are connected in series and are shown in a sequence 3 in a sequence table. In the pGPTV-SPAA, the expression of the gene of interest is controlled by the GluC promoter and the GluB5 terminator.
The microorganism may be a yeast, bacterium, algae or fungus. Wherein the bacterium can be an Agrobacterium, such as Agrobacterium EHA 105. The recombinant microorganism may be a recombinant microorganism obtained by introducing the recombinant vector into the microorganism.
The transgenic cell line, the transgenic tissue, and the transgenic organ do not include propagation material. The transgenic cell line, the transgenic tissue and the transgenic organ can be obtained by the method.
In order to solve the technical problem, the invention also provides any one of the following applications:
h1, the application of the product in preparing products for reducing animal blood pressure;
h2, the use of the product for lowering blood pressure in animals;
h3, and the application of the method in preparing products for reducing animal blood pressure.
In the present invention, the animal may be a hypertensive animal. The animal may in particular be a mammal, such as a rat or a human. The blood pressure may be systolic pressure.
Experiments prove that the transgenic plant seeds prepared by the preparation method of the plant with the function of reducing the animal blood pressure can effectively reduce the blood pressure of hypertensive animals, and the maximum reduction value is about 49.6mmHg, which is obviously higher than that of other reported methods. Therefore, the method and the protein and the gene thereof related to blood pressure reduction used in the method can be used for reducing the blood pressure of the hypertensive animals. The exogenous recombinant protein and polypeptide medicine are specifically expressed in plants by utilizing a transgenic technology, so that people can prevent and treat diseases in daily diet, and the transgenic plant can play a very important role in improving the life quality of people. Compared with the traditional fermentation technology, the transgenic plant is utilized to produce the drug protein and the drug polypeptide, and has more advantages, such as better safety, higher yield, more considerable economy and the like.
Drawings
FIG. 1 shows the structure between the LB and RB pGPTV-SPAA vectors. Wherein GluB 5T represents GluB5 terminator, GluC pro represents GluC promoter, 35S pro represents 35S promoter, and Ag 7T represents gene 7 terminator.
FIG. 2 is T0PCR detection results of transgenic pGPTV-SPAA rice.
FIG. 3 is a drawing showingT2And (3) Southern hybridization detection results of transgenic pGPTV-SPAA rice plants.
FIG. 4 is T2And (3) Northern hybridization detection results of the generation-transferred pGPTV-SPAA rice seeds.
FIG. 5 is T2Western blot detection results of the transgenic pGPTV-SPAA rice seeds.
FIG. 6 is a graph showing the quantitative determination of SPAA accumulation in pGPTV-SPAA transgenic rice. A is a Western hybridization result; b is the quantitative result of the protein of prokaryotic expression SPAA; c is the quantitative result of SPAA in the transgenic plants.
FIG. 7 is a graph showing the blood pressure changes of the rats in the WKY-C and SHR-C groups after gavage with physiological saline. ". and" represent P <0.01 and P <0.001, respectively, as compared to the WKY-C group. The blood pressure value is the value of the systolic blood pressure.
FIG. 8 shows the systolic blood pressure changes after gavage in rats W-200T and rats W-200V. The blood pressure value is the value of the systolic blood pressure.
FIG. 9 shows the systolic blood pressure changes after gavage in rats of the S-50V group and the S-50T group. ". ANG" means that P is less than 0.001 compared with the group S-C. The blood pressure value is the value of the systolic blood pressure.
FIG. 10 shows the systolic blood pressure changes after gavage in rats of the S-100V group and the S-100T group. "Tu" and "Tu" indicate that P <0.05 and P <0.01, respectively, compared to group S-C; "#" indicates that P is <0.05 compared to S-100V. The blood pressure value is the value of the systolic blood pressure.
FIG. 11 shows the systolic blood pressure changes after gavage in rats of the S-200V group and the S-200T group. "," and "" indicate that P <0.05, P <0.01 and P <0.001, respectively, compared to the S-C group; "#" and "# # #" indicate that P <0.05 and P <0.001, respectively, as compared to S-200V. The blood pressure value is the value of the systolic blood pressure.
FIG. 12 shows the systolic blood pressure changes of rats in the W-200V group and the W-200T group. "+ 1" and "+ 2" indicate the first and second weeks, respectively, after cessation of rice flour feeding.
FIG. 13 shows the systolic blood pressure changes in rats of the S-50V group and the S-50T group. "+ 1" and "+ 2" indicate the first and second weeks, respectively, after stopping feeding the rice flour; "Tuo" indicates that the difference reaches a P <0.05 level.
Detailed Description
The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The experimental procedures in the following examples are conventional unless otherwise specified. Materials, reagents, instruments and the like used in the following examples are commercially available unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged. In the following examples, unless otherwise specified, the 1 st position of each nucleotide sequence in the sequence listing is the 5 'terminal nucleotide of the corresponding DNA, and the last position is the 3' terminal nucleotide of the corresponding DNA.
The rice (Oryza sativa cv Kitaake) ((Wen tying Li. Ling Dai. Zhi Jian Chai. Zhi Jie Yin. Le Qi. Long Qi. evaluation of seed storage protein gene 3' -untranslated regions in enhancing gene expression in transforming protein seed. Transmission Res (2012)21: 545. 553.) in the following examples is publicly available from the applicant, and is only used for repeating the experiments related to the present invention, but is not used for other purposes.
Example 1 preparation of blood pressure lowering related protein and Gene encoding the same
The present example provides a protein with blood pressure lowering function obtained by connecting ten Angiotensin Converting Enzyme Inhibitory Peptides (ACEIPs), which is named as a blood pressure lowering related protein (SPAA), the ACEIPs used have the sequences of 3 rd to 8 th, 11 th to 16 th, 19 th to 30 th, 31 th to 33 th, 34 th to 40 th, 43 th to 47 th, 50 th to 61 th, 64 th to 66 th, 68 th to 73 th and 76 th to 85 th of sequence 1 in the sequence table, two ACEIPs which cannot be digested by pepsin are connected by glutamine and arginine, so that two polypeptides which cannot be digested by pepsin can be digested by pancreatin, and the amino acid sequence of SPAA is sequence 1 in the sequence table.
The DNA fragment is marked as SPAA gene according to the nucleotide sequence artificially designed and coded by the rice codon preference, the sequence is the 106 th-360 th position of the sequence 2 in the sequence table, and the SPAA gene is synthesized by the biological engineering (Shanghai) company Limited.
Example 2 preparation of novel antihypertensive transgenic rice material and detection of antihypertensive function thereof
Construction of one-element and two-element plant expression vector
Artificially synthesizing a DNA molecule shown as a sequence 2 in the sequence table, wherein the 1 st to 105 th sites of the sequence 2 are GluA2 signal peptide sequences, the 106 th and the 361 th and the 615 th sites are SPAA gene sequences, and a DNA fragment shown as the sequence 2 is marked as GluA2-2SPAA gene.
Replacing a DNA fragment between recognition sequences of SmaI and SacI of pGPTV-pGluc-tGluB5 expression vector (Wen tying Li. Ling Link Dai. Zhi Jian Chan. Zhi Jie Yi. Le Qi ng Qu.evaluation of cut storage protein gene 3' -transformed genes in enhancing gene expression in transforming protein sequence (2012)21: 545. 553. the expression of the GluA 2-2A gene in the recombinant vector is controlled by a GluC promoter (SEQ ID NO: 3) and a GluB terminator (SEQ ID NO: 83. the expression of GluB terminator) in the GluC promoter (SEQ ID NO: 5. the expression of GluC 2-2A gene in the recombinant vector is controlled by a plasmid DNA promoter (SEQ ID NO: 35. the expression of GluC promoter and plasmid promoter) in the plasmid vector strain expression vector (SEQ ID NO: 5. the expression of GluC promoter) 20:1195-1197, 1992) for expression of the hygromycin resistance gene. The structure between the LB and RB pGPTV-SPAA vectors is shown in FIG. 1.
Wherein, the 1 st to 35 th sites of the sequence 3 are GluA2 signal peptide, and the 36 th to 120 th sites and the 121 th and 205 th sites are SPAA.
Second, construction of transgenic pGPTV-SPAA plant expression vector rice
And (3) introducing the pGPTV-SPAA obtained in the first step into the agrobacterium EHA105 by a freeze-thaw method, and then transforming rice ktia-ake embryogenic callus by an agrobacterium-mediated method. 24 strains of T were obtained by hygromycin screening0Transgenic plants are generated. Introducing the pGPTV-pGluc-tGluB5 expression vector into agrobacterium EHA105, and then transforming rice ktia-ake embryonic callus by an agrobacterium-mediated method to obtain empty cellsThe vector was rice.
Molecular detection of transgenic rice plant
1、T0PCR detection of generation-transferred pGPTV-SPAA rice
T-PCR method0And (5) detecting plant molecules. The hygromycin gene primer sequences used for detection are as follows:
Hpt-F:5'AGTTCGACAGCGTCTCCGACCTG 3'
Hpt-R:5'CCTGCGCCCAAGCTGCATCATCG 3'
the target band of 819bp obtained by PCR amplification is the positive detection. In the experiment, non-transgenic rice Kitaake is set as a negative control, and pGPTV-SPAA plasmid is used as a positive control.
The results show that: the obtained 24 strains of T0All the transgenic plants were positive (FIG. 2). In the figure, M is a DNA molecular weight standard, + is a positive control, NT is a negative control, and Q1-Q24 are 24 strains of T respectively0Transgenic plants are generated.
2、T2Southern hybridization detection of transgenic pGPTV-SPAA rice plants
24 strains of T0Inbreeding of transgenic plants to obtain T2Transgenic plants are generated. Extraction of T by CTAB method2The genomic DNA of the transgenic plant leaf was digested by adding SacI to about 25. mu.g of the genomic DNA for 24 hours, and the resulting digested product was separated by 0.8% agarose gel electrophoresis and transferred to a nylon membrane with positively charged nuclei (Amersham pharmacia). Hybridization and signal detection of the nylon membrane and the probe were carried out with reference to the Roche II type high-efficiency digoxin DNA labeling and detection kit (cat # 11585614910). The hygromycin gene fragment obtained by PCR amplification of the hygromycin gene by using Hpt1 and Hpt2 is used as a hybridization probe, and the primer sequences are as follows:
Hpt1：5’-GCAAGGAATCGGT-CAATACA-3’
Hpt2：5’-TTCTACACAGCCATCGGTC-3’
the Southern hybridization results showed that the hybridizing bands of the hygromycin gene were detected in all 8 transgenic lines and that the number and the position of appearance of the hybridizing bands were not identical in the different lines, which is shown in the tableThe SPAA gene has been successfully integrated into the rice genome, and independent transformed individuals existed between the transgenic lines (FIG. 3). In the figure, kita is rice ktia-ake as a non-transgenic plant control, and Q1-Q8 are T0T with the number of Q1-Q8 in generation transgenic plants2And (5) plant generation.
3、T2Northern hybridization detection of generation-transferred pGPTV-SPAA rice seeds
Total RNA from transgenic rice seeds 10 days after flowering was extracted with TRIpure reagent (Baitach), about 25. mu.g of the total RNA was separated by 0.8% agarose gel electrophoresis, and the RNA was transferred to a nylon membrane with positively charged nuclei (Amersham pharmacia) using 20 XSSC. Hybridization and signal detection of the transferred membrane and probe were carried out with reference to the Roche II type efficient digoxin DNA labeling and detection kit (cat # 11585614910). The full-length nucleotide sequence of the SPAA gene was used as a hybridization probe.
Northern hybridization results showed that the SPAA gene was expressed to a different extent at the transcriptional level in pGPTV-SPAA transgenic rice seeds compared to the non-transgenic control plants (rice ktia-ake) (FIG. 4). In the figure, kita is non-transgenic plant control, Q2, Q3, Q6 and Q8 are T respectively0T with the numbers of Q2, Q3, Q6 and Q8 in generation transgenic plants2And (5) plant generation. This indicates that in transgenic plants, the GluC promoter and GluB5 gene terminator can effectively and specifically drive and regulate the expression of SPAA gene in seed endosperm at the transcription level.
4、T2Western hybridization detection of generation-transferred pGPTV-SPAA rice seeds
From T2Total protein was extracted from mature seeds of rice transformed with pGPTV-SPAA, the seeds were sufficiently ground, an extraction buffer (formulation: 0.125M Tris-HCl,4M urea, 4% SDS and 2% beta-mercaptoethanol, pH 6.8) was added thereto, the mixture was shaken overnight, and then centrifuged at 12000g for 5min, the supernatant was collected, and after electrophoresis with 13.6% SDS-PAGE gel, the protein was transferred onto a PVDF membrane by a semidry method (Millipore Co.). The membrane is sequentially subjected to sealing, rinsing, primary antibody, rinsing, secondary antibody, rinsing, ECL hypersensitive luminescent solution reaction (Amersham Biosciences), tabletting, developing, fixing and scanning to obtain a non-transgenic plantRice Kitaake was used as a control.
The preparation method of the primary antibody is as follows: the antigenicity and hydrophobicity of the SPAA amino acid sequence are analyzed by using DNAman software, and the sequence 3 in the sequence table is selected as an antigen sequence. Constructing a nucleotide sequence (sequence 2 in a sequence table) corresponding to the sequence on a prokaryotic expression vector pET-32a by utilizing enzyme cutting sites EcoRI and Xho I, expressing GluA2-2SPAA fusion His-labeled fusion protein, purifying the expressed fusion protein by utilizing a nickel column, immunizing a chicken, and obtaining immune serum as a primary antibody for Western hybridization.
The results of Western blot showed that SPAA protein was expressed to a different extent in the transgenic lines compared to the non-transgenic plant controls (FIG. 5). In the figure, kita is a non-transgenic plant control, and Q1-Q8 are T0T with the number of Q1-Q8 in generation transgenic plants2And (5) plant generation.
Four, T4Substitution pGPTV-SPAA rice seed feeding SHR blood pressure reduction detection
1. Quantitative determination of SPAA accumulation in pGPTV-SPAA transgenic rice
Selecting four T4Transferring seeds of pGPTV-SPAA rice seed strains Q2, Q3, Q6 and Q8, respectively extracting the whole protein of the rice seeds, and carrying out Western hybridization analysis according to the method in the third step. The loading amount of the proteins of the four strains is 80 mu g, the GE Healthcare protein quantitative kit (cargo number: 80-6483-56) is used for protein quantitative determination, and the specific operation is shown in the specification. Meanwhile, 0ng, 25ng, 50ng, 75ng and 100ng of prokaryotic expression SPAA protein is taken as a standard, and online software NIH ImageJ (National Institutes of Health, ver.1.41,https://www.nih.gov/)the optical density value is measured and a standard curve is drawn. Finally, the SPAA content of the four strains Q2, Q3, Q6 and Q8 was calculated (FIG. 6).
The preparation method of the prokaryotic expression SPAA protein comprises the following steps: DNA molecules shown in a sequence 2 in a sequence table are constructed on a prokaryotic expression vector pET-32a by utilizing enzyme cutting sites EcoRI and Xho I, then the DNA molecules are introduced into escherichia coli for protein expression, expressed GluA2-2SPAA fusion His-labeled fusion protein is expressed, and the expressed fusion protein is purified by utilizing a nickel column.
The results show that SPAA protein is stably expressed in four homozygous transgenic lines Q2, Q3, Q6 and Q8, and the content of SPAA protein in each 80 mu g of seed holoprotein is 27ng, 23ng, 30ng and 38ng respectively. Because about 160mg of holoprotein can be extracted from 1g of rice seeds, the SPAA protein which can accumulate 76 mu g at most in 1g of rice seeds in the strain Q8 with the highest expression quantity can be deduced.
2. Preparing rice seed protein powder:
will T4The method comprises the following steps of extracting proteins from seeds of a generation-transfer pGPTV-SPAA rice seed strain Q8, rice kitaake and empty vector control rice according to the following method, and respectively obtaining transgenic rice seed protein powder, common rice seed protein powder and empty load control rice seed protein powder:
fully grinding rice seeds, adding an extraction buffer solution (formula: 0.125M Tris-HCl,4M urea, 4% SDS and 2% beta-mercaptoethanol, pH 6.8), oscillating overnight, centrifuging for 10min at 12000g, collecting supernatant, adding precooled acetone with 6 times of volume, reversing and uniformly mixing, standing at-20 ℃ for 1h, centrifuging for 10min at 4000g, collecting precipitate, blow-drying by a nitrogen blower, fully removing acetone, grinding protein into powder by a grinder, and obtaining rice seed protein powder for later use.
3. Effect of pGPTV-SPAA Rice seed protein on rat blood pressure
The experimental animal model is selected from male 18-week-old spontaneous hypertension rats (SHR, Beijing Wintolite laboratory animal technology Co., Ltd.) and male 18-week-old SPF-level WKY rats (from Beijing Wintolite laboratory animal technology Co., Ltd.) as experimental control. Randomly grouping the WKY rats, wherein each group comprises 6-7 rats; SHR rats are randomly grouped, and 6-7 rats are in each group. The rice seed protein powder is fed in a short time by adopting an oral administration mode, the rice seed protein powder is dissolved by using normal saline, the amount of the gavage protein powder and the volume of the solution of each rat are equal, and the blood pressure changes of 2h, 4h, 6h and 8h before and after feeding are recorded. After calculating the mean systolic pressure of each rat at different time points, the mean systolic pressure of each group of rats at different time points was calculated. Data are presented as Mean ± SEM, and data analysis using Two-way ANOVA indicates significant differences with P values less than 0.05. Groups of rats were tested for systolic arterial pressure using a small animal noninvasive blood pressure analysis system (BP-2000, Visitech Systems, USA). During measurement, the rats are placed in a fixer, the temperature of a hot plate is set to be 39 ℃, after standing for 10min, the blood pressure is measured for 15 times, and the average value of all effective systolic pressures of each rat is calculated to be used as the measurement result of the rat.
The systolic pressure of the rats in the control groups of WKY and SHR rats (W-C group and S-C group) at 0h, 2h, 4h, 6h and 8h after the administration of the gastric saline is detected, and the result is shown in FIG. 7, the mean systolic pressure of the rats in the WKY-C group is about 150mmHg at each time point, and the mean systolic pressure of the SHR rats is 200mmHg, which have significant differences (P <0.0001), which indicates that the SHR rats have developed a hypertension stage and can be used as a hypertension model for subsequent experiments.
Compared with the WKY rats (W-200T group) with the same amount of intragastric administration of the transgenic rice seed protein powder (the intragastric administration amount of SPAA is 200 mug/kg body weight), the contraction pressure of the WKY rats (W-200V group) has no obvious difference after the intragastric administration for 0h, 2h, 4h, 6h and 8h (figure 8), which shows that the transgenic rice seed protein powder has no influence on the contraction pressure of the WKY rats.
The systolic pressure of SHR rats (S-50T group) of the intragastric transgenic rice seed protein powder (the intragastric amount of SPAA meets the intragastric amount of 50 mug/kg body weight) is reduced after 2h of intragastric administration, reaches the minimum value after 8h of intragastric administration, is obviously lower than that of SHR rats (P <0.001) of the intragastric physiological saline group (S-C group), and the maximum reduction value is about 49.6mmHg (figure 9). The SHR rats (S-50V group) with the same amount of protein powder/kg weight of common rice seeds after gastric lavage have no influence on the systolic arterial pressure of the SHR rats after gastric lavage for 0h, 2h, 4h, 6h and 8 h.
The systolic pressure of SHR rats (S-100T group) of the intragastric transgenic rice seed protein powder (the intragastric amount of SPAA is 100 mug/kg body weight) is obviously reduced 2h after intragastric administration (compared with S-C group, the significance analysis P is less than 0.05), but is temporarily increased 4h after intragastric administration; and the systolic blood pressure decreased continuously 6h after gavage (significance analysis P <0.01 compared with the S-C group; significance analysis P <0.05 compared with the S-100V group) and continued until 8h after gavage (significance analysis P <0.01 compared with the S-C group) (FIG. 10). The SHR rats (S-100V group) with the same amount of protein powder/kg weight of common rice seeds after gastric lavage have no influence on the systolic arterial pressure of the SHR rats after gastric lavage for 0h, 2h, 4h, 6h and 8 h.
The systolic pressure of SHR rats (S-200T group) of the intragastric transgenic rice seed protein powder (the intragastric amount of SPAA is 200 mug/kg body weight) is obviously reduced (P is less than 0.05) 4h after intragastric administration, and is continued until 8h after intragastric administration, and the minimum value is reached (compared with S-C group, the significance analysis P is less than 0.001; compared with S-200V group, the significance analysis P is less than 0.001) (figure 11). The SHR rats (S-200V group) with the same amount of protein powder/kg weight of common rice seeds after gastric lavage have no influence on the systolic arterial pressure of the SHR rats after gastric lavage for 0h, 2h, 4h, 6h and 8 h.
Equal amount of no-load control rice seed protein powder is respectively used for gavage of WKY rats and SHR rats, and the result shows that the systolic pressure has no significant change compared with the WKY rats and the SHR rats of the gavage of the common rice seed protein powder. In addition, the systolic pressure of the WKY rat and the SHR rat which are filled with the equal volume of the physiological saline is not obviously changed compared with the WKY rat and the SHR rat which are filled with the common protein powder.
The results show that the transgenic rice seed protein powder has no influence on the systolic pressure of a common rat, but can reduce the systolic pressure of a hypertensive rat, namely the SPAA transgenic rice has the function of reducing the blood pressure.
4. Effect of pGPTV-SPAA Rice seed Dry powder on rat blood pressure
Male 19-week-old Spontaneous Hypertensive Rats (SHR) were selected as experimental animal models, and male 19-week-old SPF-grade WKY rats were selected as experimental controls. Randomly grouping the WKY rats, wherein each group comprises 6-7 rats; SHR rats are randomly grouped, and 6-7 rats are in each group. Rats were weighed once a week. One week prior to the long-term experiment, each rat was gavaged with 3ml of Normal Saline (NS) once a day. During the long-acting experiment, rats dissolved the required amount of the transgenic rice flour of step 2 in 3ml of physiological saline according to their body weight of one week before, and were gavaged once a day for 5 weeks. After 5 weeks of gavage, the rats were again fed normally for two weeks. Groups of rats were tested for systolic arterial pressure using a small animal noninvasive blood pressure analysis system (BP-2000, Visitech Systems, USA). During measurement, the rats are placed in a fixer, the temperature of a hot plate is set to be 39 ℃, after standing for 10min, the blood pressure is measured for 15 times, and the average value of all effective systolic pressures of each rat is calculated to be used as the measurement result of the rat. The average systolic pressure of all rats was measured once every thursday and friday, the blood pressure value of 0h of the short-term test was taken as the blood pressure 0 point, and the difference (Δ P) between the systolic pressure and the blood pressure 0 point of each group was calculated and plotted with the difference as the vertical axis and time as the horizontal axis. Data are presented as Mean ± SEM, and data analysis using Two-way ANOVA indicates significant differences with P values less than 0.05.
The mean systolic arterial pressure of the WKY rats was measured once a week, and the difference in blood pressure (. DELTA.P) was calculated based on the 0h blood pressure value of the short-lived experiment. As shown in FIG. 12, WKY rats (W-200T group) with continuous gavage of transgenic rice flour (the gavage amount of SPAA is 200 mug/kg body weight) have no influence on the arterial systolic pressure of the WKY rats, and have no significant difference compared with the same amount of ordinary rice flour WKY rats (W-200V group) in gavage; the contraction pressure of WKY rats with the same amount of the control rice flour after gastric lavage and the contraction pressure of the WKY rats with the common rice flour have no significant difference. The systolic pressure of SHR rats (S-50T group) with the gavage transgenic rice flour (the gavage amount of SPAA meets the requirement that the gavage amount is 50 mug/kg body weight) is obviously reduced 2 weeks after gavage (P is less than 0.05), and is lowest 5 weeks after gavage, and the SHR rats have obvious difference compared with the SHR rats (S-50V group) with the same amount of common rice flour (fig. 13); the shrinkages of SHR rats with the same amount of the control rice flour, the common rice flour and the physiological saline water have no obvious difference. The transgenic rice flour can reduce the systolic pressure of a hypertensive rat, and the transgenic pGPTV-SPAA rice has the function of reducing the blood pressure.
Wherein, rice obtained by shelling rice kitaake seeds is ground into powder, namely common rice powder; will T4Rice obtained after seeds of a generation transfer pGPTV-SPAA rice seed strain Q8 are hulled is ground into powder, and the powder is transgenic rice powder; and grinding the rice obtained after hulling the empty carrier control rice seeds into powder, namely the control rice powder.
<110> institute of plant of Chinese academy of sciences
<120> preparation method of blood pressure-reducing rice material, and gene and protein used in preparation method
<160> 5
<170> PatentIn version 3.5
<210> 1
<211> 85
<212> PRT
<213> Artificial sequence
<220>
<223>
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Gln Arg Asp Lys Ile His Pro Phe Gln Arg Tyr Gln Gln Pro Val Leu
1 5 10 15
Gln Arg Phe Phe Val Ala Pro Phe Pro Glu Val Phe Gly Lys Ile Pro
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Pro Lys Val Leu Pro Val Pro Glu Gln Arg Leu Lys Pro Asn Met Gln
35 40 45
Arg Tyr Leu Ala His Lys Ala Leu Pro Met His Ile Arg Gln Arg Val
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Phe Pro Gly Pro Ile
85
<210> 2
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<212> DNA
<213> Artificial sequence
<220>
<223>
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atggcatcca taaatcgccc catagttttc ttcacagttt gcttgttcct cttgtgcgat 60
ggctccctag ccgtatatat catccaaggg agaggtataa cagggcaacg tgataagatt 120
catcccttcc agcgttacca gcaaccagtt ctacagagat tcttcgttgc cccatttcca 180
gaagtctttg gaaagattcc acctaaggtt ctcccagtac ccgagcagag gcttaagcca 240
aatatgcaac gttatcttgc acataaggcc ttgccaatgc acatccggca aagggttccc 300
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caacgtgata agattcatcc cttccagcgt taccagcaac cagttctaca gagattcttc 420
gttgccccat ttccagaagt ctttggaaag attccaccta aggttctccc agtacccgag 480
cagaggctta agccaaatat gcaacgttat cttgcacata aggccttgcc aatgcacatc 540
cggcaaaggg ttccccctca acgtccactt aagccttggc agcgtagtaa ggtctaccca 600
tttccgggtc caatc 615
<210> 3
<211> 205
<212> PRT
<213> Artificial sequence
<220>
<223>
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Met Ala Ser Ile Asn Arg Pro Ile Val Phe Phe Thr Val Cys Leu Phe
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Leu Leu Cys Asp Gly Ser Leu Ala Val Tyr Ile Ile Gln Gly Arg Gly
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Ile Thr Gly Gln Arg Asp Lys Ile His Pro Phe Gln Arg Tyr Gln Gln
35 40 45
Pro Val Leu Gln Arg Phe Phe Val Ala Pro Phe Pro Glu Val Phe Gly
50 55 60
Lys Ile Pro Pro Lys Val Leu Pro Val Pro Glu Gln Arg Leu Lys Pro
65 70 75 80
Asn Met Gln Arg Tyr Leu Ala His Lys Ala Leu Pro Met His Ile Arg
85 90 95
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Gln Arg Leu Lys Pro Asn Met Gln Arg Tyr Leu Ala His Lys Ala Leu
165 170 175
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180 185 190
Trp Gln Arg Ser Lys Val Tyr Pro Phe Pro Gly Pro Ile
195 200 205
<210> 4
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<213> Rice (Oryza sativa L.)
<400> 4
gttcaagatt tatttttggt atttaattta cttgcttaag tcagatatat tcccatcgtt 60
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gaaagtttat tcacgaggct agagtagtaa tcaataacat aagcgtggtg tctaggtcag 180
cggttatctt catatgtagt gtgctccatg gaaagtgagg taggaggaag gtggtgacag 240
tcccgtccgt cctttgtatc cctccatgtt cgggtatatc atagagctac aggctagact 300
tagcttggca gactagggga gagccggtgc tcgaagcaat ccatgaggct ttacatttaa 360
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cttggatata attatattcc tacatataca tacacgttcc ctgcgattag atacccttgg 480
aatactctaa ggtgaagtgc tacagcggta tccgtgcgct tgcggattta tctgtgaccg 540
tatcaaatac caacaggtag atacaaggaa tcatctctcc tatccattgg tttatcatct 600
tttaaaatta tctcttgctc tcctattgcc tctgcaactg cggataggtg tttctcaaca 660
atgaaggttg tgaagaatgc tttgtgcaac aagatggatg acaagtatct cagccatagc 720
ctcatttgct ttgtagaaaa ggatatgtcg gacacaatca ctaagtatca ccgtggaaag 780
gatgcactgt atgccctatc tatatttacc atttagtaat atttatatgg cttgtgctaa 840
ctttatgttg tctttacagg caataacatt atttggaagg catatctata tattactatt 900
taagataatg taatatctca aagtttttat aagctgcaat gaggtgagtt tcacttagct 960
ttctaacttg ttatgagtta tagatgcatg ccaccagtca ttttttatct tgcatcagcc 1020
cctgcctgtt agaatatgtt tctttgtctg ggagtccatg tcaactagcc aatttccaaa 1080
tatatgaaca aaactatgtg gcctttgtaa cccaaatgag ataaagacta ctctccatag 1140
aaatttagca aacatggcac tcaaagaaaa tgtgttggat agtttcatca tgcatacaaa 1200
agcaacactt ttgaactacc attccaaatc ctttttgtaa attatctttg cttaacacta 1260
cccctttgag caaatgtggc tttgtgcgga aaaaactcaa acttggtagg gtagacatcc 1320
atttatataa ttggatccat gtacataagt tgttgagtac ttcaagtact tacccttgtg 1380
atatacatct caaatatatt gaagaagaga agttcttttt ttgagagagg ttgaagaaga 1440
gaagtttgtc catagctgaa gaggagtttt atagtgtcta gcttaccttg ctgctgattg 1500
catgtctaaa atgtcgttta atttgggcta taatgaaata ttcaccaata tttctgctgg 1560
tctattaaag tttaatagtt actcgtaact catttatttt gggctataat ttaatattca 1620
cctatgtttt tgttagtcta ttttatttcc ctagtgtgca ctagcttaac cccaaattag 1680
ttttgaacac ttaacctaaa tgtgtctatt atggtcagac actctctcac ggcactctaa 1740
caaaaagtga attttgttgt tatgtttttg tcatgatctc acaagcaatg tacatgtacg 1800
tttctagagt gcaatcttat gctagcctga ttgtgaattt agtgtagttt gttttctctt 1860
tttgtagcta cactaccaat aacctattgt cctctagtca taccacgtaa tcacaaggca 1920
aatccctaac tctcaccttt aaaagcatgt ctttattttc ttgggtggca ctaatacaaa 1980
atctttttca gcattcctat gtgcgatagc aagaaaacat ggcataactc ttgcttcact 2040
ctaacaaaaa aaacactttt ccaactttaa aacaatggta tctatgtgtt taatgatcaa 2100
tcaagcatat aatgacttac aagtttttac ctatgccctt tttgcatcat cttgtttgca 2160
acagacaaac tagatattcc tttaggctat aaacacatca gcatgataaa gagattaggt 2220
aagtttgtta tccctttttg catatattct cgtctactcc gtgtatataa gcccctctcc 2280
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<210> 5
<211> 497
<212> DNA
<213> Rice (Oryza sativa L.)
<400> 5
acccaaggca ttatatacta aaaaataaag ccacatgcat aaagctttgt ggagttttgt 60
attttgtcgt atgtgtaagt taaataaaaa aagaattgcc ttcatttaat cgatttgtgc 120
acttactccc tttatatccc atcttatttt tttctttagt aaattaaatt actcttgcct 180
ctctaaggcg cctttgtttt tagcaaaaat caacatttaa aaattgacta ctaatttaac 240
taaaatatat atgtgttacg tgttatatac tctcatacat gtaccttata ttcgtgtttt 300
aaagtaattt tatatgatac taattaagtg gttattgaca acatattaac aaaaggtcaa 360
tgataaaaag atagtgtcgg agactatgtc aaaaagttaa aattgcctta tattgtattt 420
tagaacggaa aagacgtaat ttgcatactt gcgttccatc actgcctcac ggcaaacagt 480
gtcgtggcac caagttt 497

Claims (18)

1. The preparation method of the plant with the function of reducing the blood pressure of the animal comprises the following steps: improving the expression level of the protein related to blood pressure reduction in the target plant to obtain the plant with the function of reducing the animal blood pressure;
the blood pressure reduction related protein is obtained by directly connecting or connecting ten polypeptides of the following A1) -A10):
A1) a polypeptide shown in positions 3-8 of sequence 1;
A2) a polypeptide shown in 11 th to 16 th positions of the sequence 1;
A3) a polypeptide shown in 19 th to 30 th positions of the sequence 1;
A4) a polypeptide shown in 31 st to 33 rd positions of the sequence 1;
A5) a polypeptide shown in positions 34-40 of sequence 1;
A6) a polypeptide shown in positions 43-47 of sequence 1;
A7) a polypeptide shown in positions 50-61 of sequence 1;
A8) a polypeptide shown in positions 64-66 of sequence 1;
A9) a polypeptide as shown in positions 68-73 of sequence 1;
A10) a polypeptide shown in positions 76-85 of sequence 1.
2. The method of claim 1, wherein: the blood pressure reduction related protein is B1) or B2):
B1) the amino acid sequence is the protein of sequence 1;
B2) b1) at the N-terminus or/and C-terminus.
3. The method according to claim 1 or 2, characterized in that: the improvement of the expression level of a blood pressure-lowering protein in a target plant is achieved by introducing an expression cassette containing a gene encoding the blood pressure-lowering protein according to claim 1 or 2 into the target plant.
4. The method of claim 3, wherein: the coding gene is a cDNA molecule or DNA molecule with the coding sequence of the 106 th-360 th site of the sequence 2 in the sequence table.
5. The method of claim 3, wherein: the expression of the coding gene in the expression cassette is initiated by a tissue-specific promoter capable of initiating expression of the coding gene in the edible part of the animal.
6. The method according to claim 1 or 2, characterized in that: the target plant is a monocotyledon or a dicotyledon.
7. The method of claim 6, wherein: the monocotyledon is a gramineous plant.
8. The method of claim 7, wherein: the gramineous plant is rice, corn, wheat, sorghum, oat, barley or rye.
9. The blood pressure lowering related protein according to claim 1.
10. A nucleic acid molecule encoding the blood pressure lowering related protein of claim 9.
11. An expression cassette comprising the nucleic acid molecule of claim 10.
12. A recombinant vector comprising the nucleic acid molecule of claim 10, or a recombinant vector comprising the expression cassette of claim 11.
13. A recombinant microorganism comprising the nucleic acid molecule of claim 10, or a recombinant microorganism comprising the expression cassette of claim 11, or a recombinant microorganism comprising the recombinant vector of claim 12.
14. A transgenic plant cell line comprising the nucleic acid molecule of claim 10, or a transgenic plant cell line comprising the expression cassette of claim 11.
15. A transgenic plant tissue comprising the nucleic acid molecule of claim 10, or a transgenic plant tissue comprising the expression cassette of claim 11.
16. A transgenic plant organ containing the nucleic acid molecule of claim 10, or a transgenic plant organ containing the expression cassette of claim 11.
17. Use of a blood pressure lowering protein according to claim 9, a nucleic acid molecule according to claim 10, an expression cassette according to claim 11, a recombinant vector according to claim 12, a recombinant microorganism according to claim 13, a transgenic plant cell line according to claim 14, a transgenic plant tissue according to claim 15 or a transgenic plant organ according to claim 16 for the manufacture of a product for lowering blood pressure in an animal.
18. Use of the method of any one of claims 1-8 for the manufacture of a product for lowering blood pressure in an animal.
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