CN105505946B

CN105505946B - A kind of gene GF14e, albumen and its application improving Pyricularia Oryzae resistance

Info

Publication number: CN105505946B
Application number: CN201510828431.1A
Authority: CN
Inventors: 刘清; 朱小源; 刘斌; 赵均良; 毛兴学; 王晓飞
Original assignee: Rice Research Institute of Guangdong Academy of Agricultural Sciences
Current assignee: Rice Research Institute of Guangdong Academy of Agricultural Sciences
Priority date: 2015-11-25
Filing date: 2015-11-25
Publication date: 2019-04-23
Anticipated expiration: 2035-11-25
Also published as: CN105505946A

Abstract

The invention discloses a kind of gene GF14e, albumen and its application for improving Pyricularia Oryzae resistance, the gene has the nucleotide sequence as shown in SEQ ID No:1.First demonstration that paddy gene GF14e is the functional gene of Pyricularia Oryzae resistance, the clone of the gene and biological function verification have the Study on Molecular Mechanism of Rice Resistance To Rice Blast important reference significance；The expression quantity of GF14e can be greatly improved after the rice conversion over-express vector rice transformation of overexpression GF14e gene, along with the raising of expression quantity, the rice blast neck blast resistance of transformed plant is remarkably reinforced, and transgenic plant does not change significantly on growth conditions and economical character.The technology that the present invention is overexpressed GF14e can be applied to the gene genetic Engineering Breeding of rice, and can be applied in production practices, improve the rice blast resistance of rice, to ensure the Rice Production safety under the weather conditions that current rice disease takes place frequently.

Description

A kind of gene GF14e, albumen and its application improving Pyricularia Oryzae resistance

Technical field

The invention belongs to crop genetic technical field, more particularly it relates to a kind of Pyricularia Oryzae resistance that improves Gene GF14e, albumen and its application.

Background technique

The rice blast as caused by fungi Magnaporthe grisea (Hebert) Barr is each rice region harm in the world One of rice disease of most serious makees cultivation to global rice and generates destructive influence.The whole world caused by rice blast every year because producing Amount loss is up to 11~30%, and about 5,000,000,000 dollars of direct economic loss, the grain of loss is enough to support 60,000,000 populations.In China, There is rice blast in all places for having rice cropping.South And North Rice Regions per being endangered in various degree every year, the general underproduction 10~ 20%, weight up to 40~50%, even No kernels or seeds are gathered, as in a year of scarcity for local field.

Currently, using the resistance of kind, it is considered most economical, efficient and environmentally friendly for carrying out rice blast resistance breeding Controlling way.In recent years, breeding scholars have cultivated many rice blast disease-resistant varieties using traditional breeding techniques.But It is that most of disease-resistant variety loses its rice blast resistance after promoting and applying 2~3 years.Such as high resistant to rice blast kind " narrow leaf blueness 8 Number " popularizing planting just lost resistance less than 3 years, high-yield disease resisting fine quality " round-grained rice Xian 89 " was promoted less than 4 years, disease resistance It is decreased obviously.It can be seen that the Rice Blast resistance period it is short be in Rice Production one universal and distinct issues, prolong The holding property service life of long Rice Blast, which has become, in China or even each Rice Production state rice modification to be needed preferentially to solve Problem.

Rice blast may all occur in paddy growth each period, and You Yiye pest and fringe pest are most commonly seen.Compared with leaf pest For, the harm of fringe pest is bigger, can lead to most serious when that No kernels or seeds are gathered, as in a year of scarcity.Although existing research shows that leaf blast in rice is anti- Property and neck blast resistance have intercommunity, but resistance to leaf blast and neck blast resistance also have different place.We are with 31 early period Discovery when near isogenic lines detects its leaf pest and neck blast resistance has some gene lines to have resistance to leaf blast but no neck blast resistance, instead ?.In addition, we have found when excavating the gene of leaf blast in rice and fringe pest response with biochip technology, at least 40% Gene leaf pest and fringe pest inoculation after expression pattern be different.

In summary studies have shown that leaf blast in rice resistance and neck blast resistance are different to a certain extent.Therefore, it is Solve the problems, such as that paddy disease-resistant sexual cycle is short, the neck blast resistance for solving rice also seems is even more important.However be currently known Research be essentially all for leaf blast in rice resistance, and it is then considerably less to the research of neck blast resistance.Currently, in order to improve rice Rice blast resistance, it is very urgent for the research of Pyricularia Oryzae resistance.

Summary of the invention

Based on this, in order to overcome the defects of the prior art described above, the present invention provides a kind of raising Pyricularia Oryzae resistances Gene GF14e, albumen and its application.

In order to achieve the above-mentioned object of the invention, this invention takes following specific technical solutions:

A kind of gene GF14e improving Pyricularia Oryzae resistance, the gene have the nucleotide as shown in SEQ ID No:1 Sequence.

The present invention also provides a kind of albumen for improving Pyricularia Oryzae resistance, the albumen has such as SEQ ID No:2 institute The amino acid sequence shown.

In wherein some embodiments, the albumen is as nucleotide sequence coded as shown in SEQ ID No:1.

The present invention also provides the gene GF14e for improving Pyricularia Oryzae resistance in terms of improving Pyricularia Oryzae resistance Using.

14-3-3 gene family is a kind of very important Analysis of Defence Genes Involved, plant it is disease-resistant in played important function. In rice, 14-3-3 gene family includes 8 members, i.e. GF14a-h altogether.Existing research shows that GF14e is in leaf blast in rice The regulating and controlling effect of negative sense is played in resistance.It will lead to the rice blast resistance of rice leaf with the expression of RNAi technology silencing GF14e Increase.However, the present inventor has found when excavating the gene of leaf blast in rice and fringe pest response with biochip technology, Response of the GF14e after leaf pest and fringe pest inoculation is different.After the inoculation of leaf pest, the expression of GF14e is lowered, this with before Result of study be consistent, but after fringe pest inoculation, the expression of GF14e is up-regulation, this show its in leaf pest and Opposite effect may be played in neck blast resistance.Therefore, GF14e is possible to play positive regulating and controlling effect in neck blast resistance.

Compared with prior art, the invention has the following advantages:

1. first demonstration that paddy gene GF14e is the functional gene of Pyricularia Oryzae resistance, the clone of the gene There is important reference significance with Study on Molecular Mechanism of the biological function verification for Rice Resistance To Rice Blast；

2. the present invention provides the rice conversion over-express vectors for being overexpressed GF14e gene using Camv35S promoter.It should The expression quantity that GF14e can be greatly improved after carrier rice transformation, along with the raising of expression quantity, the rice blast fringe pest of transformed plant Resistance is remarkably reinforced, and transgenic plant does not change significantly on growth conditions and economical character.Therefore, the present invention is logical The technology for crossing Camv35S promoter overexpression GF14e can be applied to the gene genetic Engineering Breeding of rice, and can be applied to In production practices, the rice blast resistance of rice is improved, to ensure that the rice under the weather conditions that current rice disease takes place frequently is raw Produce safety.

Detailed description of the invention

Fig. 1 is the Vector map of over-express vector PHQSN used in embodiment 1；

Fig. 2 is GF14e expression detection figure in the transgenic plant of embodiment 2；

Fig. 3 is that influence of the GF14e to the neck blast resistance of rice plant is overexpressed in embodiment 3, and wherein * * is indicated and compareed Compared to there are extremely significant differences.

Specific embodiment

The following examples are further illustrations of the invention, rather than limiting the invention.In the following example not It indicates specific experiment condition and method, used technological means is usually conventional means well-known to those skilled in the art.

Embodiment 1 improves the building of the gene and over-express vector of Pyricularia Oryzae resistance

(1), it takes three Huang of rice blast highly resistance kind to account for No. 2 (SHZ-2) seedling leaves positions, uses TriZol Reagent (Invitrogen company, article No. are as follows: 15596026) extract blade total serum IgE, using denaturing formaldehyde gel electrophoresis and The purity and amount of UV spectrophotometer measuring total serum IgE；

(2), the total serum IgE of 1 μ g is taken to do starting reverse transcription reaction, used reverse transcriptase is PrimeScript The step of (TAKARA company), reverse transcription reaction the operation instruction with reference to the reverse transcriptase.Using reverse transcription product as template, use Primer: F, ACGCGTCGACATATCTGGCCCAAGAGCAGTA (SEQ ID No:3)；R, GGAATTCAGGCTCACAACTGACA TACCG (SEQ ID No:4), carries out PCR amplification, and polymerase used in PCR is KOD FX (Toyobo company).Reaction system is 50uL prepares PCR reaction system according to the specification of KOD FX.Reaction condition are as follows: 94 DEG C of 5min；94 DEG C of 30sec, 57 DEG C 30sec, 68 DEG C of 80sec, 35 circulations；68℃10min.The segment of PCR amplification acquisition about 1268bp；

(3), after recycling the segment using the method for PCR product direct purification, with SalI and EcoRI to segment and PHQSN (its carrier figure is as shown in Figure 1, those skilled in the art can be with bibliography: the cDNA clone of arabidopsis AtNHX5 gene for carrier And its building of justice, antisense expression vector；Shi Leyi, Li Hongqing, Li Meiru, Chen Yizhu；Plant Physiology Communications, 2005 Year, volume 41, the 5th phase constructed and obtain) carry out double digestion respectively, 37 DEG C digestion 6 hours, be separately recovered after digestion target fragment with Carrier segments, overnight with 16 DEG C of T4 ligase (NEB company) connections, linked system are as follows: T4 ligase 1ul, 10 × buffer 1ul, GF14e genetic fragment 4ul (51ng), PHQSN carrier 2uL (50ng), aqua sterilisa 2uL；

(4), whole connection products are taken, are transformed into bacillus coli DH 5 alpha with electric shocking method, converted product is coated with kanamycins The LB solid medium of resistance.37 DEG C of overnight incubations choose 6 positive white monoclonals and extract plasmid, digestion identification, selection Two positive colonies are sequenced.Sequencing result shows: 1268 bases of the sequence, includes an open reading frame, i.e., For GF14e gene, size is 789 bases, and for nucleotide sequence as shown in SEQ ID No:1, coding albumen has 262 A amino acid residue, amino acid sequence is as shown in SEQ ID No:2.

Overexpression effect identification of the 2 GF14e gene of embodiment in transgenic plant

Over-express vector is transferred to by normal japonica rice variety OryzasativaLcv.Nipponbare using the genetic transforming method that Agrobacterium EHA105 is mediated In.Convert primary (T₀Generation) by PCR and quantitative PCR detection, positive transformants plant, card are identified from DNA level and rna level Bright target gene GF14e has been transformed into rice, and realizes that the expression quantity of target gene GF14e improves.

Transgenic positive plant selfing is obtained homozygous 1 generation of positive transgenic (T in controlled greenhouse₁Generation) strain, often A strain selects 10 plants of plant breedings through PCR test positive, obtains T₂For plant, T₂PCR detection is carried out again for strain, is looked for To from different T₀For the T of plant₂For homozygous lines 3 (5-1,12-1,12-2).The seedling leaves of 3 strains are carried out Quantitative fluorescent PCR detects the expression quantity of target gene GF14e, is control with wild-type results.

It is as follows that quantitative fluorescent PCR identification GF14e gene is overexpressed effect programs in transgenic plant:

1, the small ear for the transgenic plant beginning ear period for taking PCR detection positive carries out Total RNAs extraction, the reagent used for TriZol Reagent (Invitrogen company, article No. are as follows: 15596026), according to the step of the specification of the reagent into Row, and with the purity and amount of denaturing formaldehyde gel electrophoresis and UV spectrophotometer measuring total serum IgE；

2, the total serum IgE of 1 μ g is taken to do starting reverse transcription reaction, used reverse transcriptase is PrimeScript (Takara Company), with reference to the operation instruction of the reverse transcriptase the step of reverse transcription reaction.Using reverse transcription product as template, using primer The expression of GF14eF and GF14eR primer pair detection GF14e gene；Primer sequence is as follows: GF14eF, GATATTGCCCTGGCAGAGTTG (SEQ ID No:5)；GF14eR, GAGATATCGGAAGTCCACAGC (SEQ ID No: 6)；

3, using the expression of the EF1 α F and EF1 α R primer pair detection rice EF1 α gene of rice house-keeping gene EF1 α gene As internal reference, quantitative PCR reagent isPremix Ex Taq^TM(TAKARA), quantitative PCR instruments are CFX 96 (BioRAD company),

EF1 α F, TTTCACTCTTGGTGTGAAGCAGAT (SEQ ID No:7)；

EF1 α R, GACTTCCTTCACGATTTCATCGTAA (SEQ ID No:8).

The opposite mRNA of GF14e expresses water in 3 transgenic homozygous strains (5-1,12-1,12-2) and WT lines For reef knot fruit as shown in Fig. 2, Fig. 2 is the result shows that 3 strains are relative to wild type, the expression quantity of GF14e all increases substantially 10-40 Times.

The identification of 3 GF14e of embodiment overexpression transgenic plant rice blast neck blast resistance

3 overexpression transgenosis T homozygous in embodiment 2₂For homozygous lines (5-1,12-1,12-2) and wild type Rice paddy seed germinates at 32 DEG C, moves in the vinyl disc equipped with soil young shoot respectively after 2 days and sows, when it grew to for 3 leaf phase By seedling replanting into large plastic barrel, every barrel 4 plants of kind, each strain at least plants 10 plants.Naturally heading stage is grown in solarium It is inoculated with.The rice blast bacterial strain used is the GD08-T13 of OryzasativaLcv.Nipponbare sensitivity.

When spike of rice extracts 2~8cm or so out, goes out spike of rice with hand sowing, small pieces cotton is taken to wrap at the lower edge 1/3 of spike of rice, Then spore liquid (the 1x 10 of 2ml is drawn⁶Spores/ml it) is injected into cotton, is wrapped up outside cotton with masking foil to prevent bacterium solution Evaporation.It in solarium, sprayed water 2 minutes every 2 hours, to guarantee that humidity is conducive to spore germination.Investigation morbidity result after 3 weeks.Knot Fruit as shown in Figure 3, Fig. 3 the result shows that GF14e be overexpressed strain neck blast resistance be remarkably reinforced, compared with wild type, spike of rice main shaft Susceptible length is obviously reduced.

The embodiments described above only express several embodiments of the present invention, and the description thereof is more specific and detailed, but simultaneously Limitations on the scope of the patent of the present invention therefore cannot be interpreted as.It should be pointed out that for those of ordinary skill in the art For, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to guarantor of the invention Protect range.Therefore, the scope of protection of the patent of the invention shall be subject to the appended claims.

Claims

1. a kind of application of gene GF14e for improving Pyricularia Oryzae resistance in terms of the resistance for improving Pyricularia Oryzae.

2. application according to claim 1, which is characterized in that in the nucleotide sequence of the gene GF14e such as sequence table Shown in SEQ ID NO:1.

3. application according to claim 1, which is characterized in that it is anti-that the gene GF14e encodes a kind of raising Pyricularia Oryzae The albumen of property, the amino acid sequence of the albumen is as shown in SEQ ID NO:2 in sequence table.