CN111676220A

CN111676220A - Long-chain non-coding RNA lnc11 of poplar and application thereof

Info

Publication number: CN111676220A
Application number: CN202010434843.8A
Authority: CN
Inventors: 刘思安; 胥猛; 祁浩然; 吴玲
Original assignee: Yangzhou University
Current assignee: Yangzhou University
Priority date: 2020-05-21
Filing date: 2020-05-21
Publication date: 2020-09-18
Anticipated expiration: 2040-05-21
Also published as: CN111676220B

Abstract

The invention discloses a novel long-chain non-coding RNA lnc11 of poplar and application thereof, wherein the nucleotide sequence of the long-chain non-coding RNA lnc11 is shown as SEQ ID NO.1, and further provides an over-expression vector of the long-chain non-coding RNA lnc11 of the poplar, a multi-target point CRISPR/Cas9 knockout vector, a host of the expression vector and application of the long-chain non-coding RNA lnc11 of the poplar in the aspect of controlling the formation of adventitious roots of the poplar. The invention obtains high knockout efficiency by transferring the knockout vector of the long-chain non-coding RNA lnc11 into the poplar protoplast, and the method can be widely used for functional research of lnc RNA. In addition, the number of adventitious roots of the transgenic poplar with lnc11 provided by the invention is obviously reduced after overexpression. The method shows that lnc11 is a key gene for regulating the formation of adventitious roots of poplar, and poplar with different rooting numbers can be cultivated as required by regulating the number of the adventitious roots of poplar through overexpression or knockout of lnc11, so that the method has important application value in the molecular breeding process of poplar.

Description

Long-chain non-coding RNA lnc11 of poplar and application thereof

Technical Field

The invention belongs to the technical field of plant genetic engineering, and particularly relates to a novel long-chain non-coding RNAlnc11 of poplar and application thereof.

Background

The poplar root system mainly comprises a large number of adventitious roots and lateral roots thereof, is the main organ for fixing, absorbing nutrients and moisture, and determines the growth, yield, resistance and ecological effect of poplar. Generally, poplar is suitable for asexual propagation, and among various asexual propagation methods, cuttage is the most common, so that the method is simple and easy to implement, high in efficiency and low in cost, and can maintain the excellent characters of the mother plant, and can blossom and fruit early. The difficulty of the generation of the adventitious roots of the cutting directly influences the survival rate of afforestation and is concerned with the adaptability and the stress resistance of the nursery stock. The difficulty in rooting by cutting of part of excellent genotypes is a main factor for limiting the popularization and afforestation of the excellent genotypes in difficult places such as drought, cold, saline-alkali tidal flats and the like.

Plasticity of adventitious root generation and root system response to adversity stress is the leading edge and hot spot of the field of plant development and evolution biology. Although research on root development biology of herbaceous plants such as arabidopsis, rice and corn has made breakthrough progress, many molecular regulation modes and signaling pathways have been revealed. However, the organ development and anti-adversity mechanism of perennial forest trees and herbaceous plants are obviously different. Deeply analyzing the genetic regulation mechanism of poplar adventitious root generation has important theoretical significance for revealing the dissimilarity of organogenesis and adaptive evolution mechanism among species. In recent years, researchers have conducted research on the excavation and functional verification of mRNA and miRNA in more and more tree species. The functional genomics research of the poplar is ahead of other tree species, and some key mRNA, miRNA and regulation and control passages thereof in the adventitious root generation process of the poplar are preliminarily verified and explained, but the formation and development of the adventitious root of the forest tree are rarely regulated by long-chain non-coding RNA (lncRNA).

IncRNA refers to non-coding RNA with length larger than 200nt, no long open reading frame and no coding capacity. Unlike mirnas, which have been studied more thoroughly, lncRNA was initiated later. Originally, lncRNA was not appreciated and was once considered to be a nonfunctional transcriptional "noise". However, since the first lncrnas reported by Lukiw et al (1992) in humans, a recent series of studies have shown that lncrnas play a major role in many life activities and developmental processes. In recent years, with the development of high-throughput sequencing technology, more and more lncRNA is predicted in plants, but there are few reports on intensive research on cloning and function of lncRNA. Although the research on the plant lncRNA is just started, the existing research finds that the biological functions of the plant lncRNA are rich and diverse, and the plant lncRNA is widely involved in the aspects of plant life activities.

In Arabidopsis thaliana, lncRNA COOLAIR is the natural antisense transcript of the key flowering inhibitor gene FLC, while COLDAIR is transcribed from the first intron of FLC, COOLAIR transiently inhibits FLC expression by transcriptional interference, and COLDAIR regulates Arabidopsis flowering by participating in chromatin appearance modification to silence FLC expression, as previously described. Inspiring and the like by comparing a wild rice Nongken 58N with a rice photosensitive male sterile line Nongken 58S, finding that the rice specific lncRNA LDMAR generates C-G single base mutation to cause methylation of a promoter region, and inhibiting the expression of the LDMAR in the Nongken 58S to cause the generation of photosensitive male sterility. The lncRNA Zm401 specifically expressed by the maize pollen is a key gene for regulating the development of the pollen, and the maize male sterility can be caused by deletion and over-expression of the Zm 401. The lncRNA BcMF11 specifically expressed by the Chinese cabbage pollen plays an important role in the fertility aspect of the Chinese cabbage, and if the expression of the BcMF11 is abnormal, the tapetum is degraded in advance and pollen abortion is caused. Woody plants are much less frequently studied about lncrnas than herbaceous plants.

Although the high-throughput sequencing work of the lncRNA of the poplar is carried out at present, few research reports for deeply researching the function of the lncRNA are reported, and particularly, the research on regulating the adventitious root of the poplar by the lncRNA is not reported.

Disclosure of Invention

The purpose of the invention is as follows: aiming at the defects in the prior art, the invention aims to provide a novel long-chain non-coding RNA lnc 11. The invention also aims to provide application of the novel long-chain non-coding RNA lnc11 in regulation and control of the formation process of adventitious roots of poplar. According to the invention, a new lncRNA-lnc 11 function is cloned and researched, so that the blank of the lncRNA in regulation and control of poplar adventitious root formation can be filled, and the research progress of forest molecular breeding can be promoted.

The technical scheme is as follows: in order to achieve the purpose of the invention, the technical scheme adopted by the invention is as follows:

the invention provides a novel poplar long-chain non-coding RNA lnc11, the nucleotide sequence of which is shown in SEQ NO. 1.

The invention also provides an overexpression vector of the poplar long-chain non-coding RNA lnc 11.

As an embodiment:

the overexpression vector is assembled at the 5' end of lnc11 to form a strong expression promoter P35S.

The overexpression vector assembles a strong terminator NOS at the 3' end of lnc 11.

The over-expression vector is assembled with an NPT II gene expression cassette, and can be used as a screening marker of transgenic poplar, and kanamycin can be used for screening the transgenic poplar.

The over-expression vector assembles LB and RB sequences, and facilitates the gene expression frame and the screening marker gene NPT II assembled between the sequences to be integrated into the chromosome of the poplar receptor cell.

The invention also provides a multi-target CRISPR/Cas9 knockout vector of the lnc 11.

As an embodiment:

the CRISPR/Cas9 knockout vector is a CRISPR/Cas9-lnc11 vector, contains an NPTII resistance gene, has kanamycin resistance, and is driven by a P35S promoter through a Cas9 protein; the target site sequence of CRISPR/Cas9 of the lnc11 is TAAACGCTGGCATGGAAAAG; GTTTATGGTTGAGTAAACGC, respectively; TCTTGAAA TGAATGTTGTAG are provided.

The invention also provides a host cell containing the long-chain non-coding RNA lnc11 of the poplar, the overexpression vector or the multi-target CRISPR/Cas9 knockout vector of lnc 11.

The invention finally provides application of the long-chain non-coding RNA lnc11 in regulation and control of poplar adventitious root formation.

The invention takes Nanlin 895 poplar adventitious roots as a material, and clones lnc11 gene. Meanwhile, the overexpression vector 35S of the gene is constructed by adopting Gateway cloning technology, i.e. lnc 11. A multi-target knockout vector CRISPR/Cas9-lnc11 is constructed by adopting overlapping PCR and gold-gated cloning technology, and high knockout efficiency is obtained by expression in poplar protoplasts. The over-expression vector 35S is transferred into poplar to find that the over-expression vector regulates the formation of poplar adventitious roots.

Has the advantages that: the invention clones a new long-chain non-coding RNA lnc11, obtains high knockout efficiency by transferring the knockout vector of the long-chain non-coding RNA lnc11 into poplar protoplast, and can be widely used for functional research of lnc RNA. The number of adventitious roots of the transgenic poplar over expressing lnc11 is obviously reduced, and the lnc11 is important lncRNA for regulating and controlling the formation of the adventitious roots of the poplar. The number of the adventitious roots of the poplar is regulated by over-expressing or knocking out lnc11, and the method has important application value in the molecular breeding process of the poplar.

Drawings

FIG. 1 is a schematic structural diagram of a constructed plant expression vector 35S, lnc 11;

FIG. 2 is CRISPR/Cas9 knockout efficiency assay of lnc 11;

FIG. 3 is the detection of transgenic poplar molecules overexpressing lnc 11;

FIG. 4 is a graph comparing the overall morphology of transgenic and non-transgenic poplars overexpressing lnc 11; CK in the figure is not transgenic poplar;

FIG. 5 is an adventitious root count statistic of transgenic and non-transgenic poplars overexpressing lnc 11.

Detailed Description

The technical solution of the present invention is further described in detail by the following specific examples.

EXAMPLE 1 clone lnc11

(1) Primers were designed using Oligo 6 based on poplar genome and transcriptome data of applicants' prophase poplar adventitious roots.

Wherein the lnc11 forward primer is 5 '-TCACTATTTATTTCCCATCAAGAAA-3' and the lnc11 reverse primer is 5 '-TGAGATTTTATCTGGGTTGTTGGT-3' and the high fidelity PCR reaction system is 10 × LA PCR Buffer (Mg)²⁺free)5.0μl；2.5mM dNTP Mixture 8.0μl；25mM Mg²⁺5.0 μ l; LA Taq DNA Polymerase (5U/. mu.l) 0.5. mu.l; 2. mu.l of forward primer (10. mu.M); reverse primer (1)0 μ M)2 μ l; template (895 poplar cDNA) 1. mu.l; add sterile ddH₂O complement 50. mu.l reaction program pre-denaturation at 94 ℃ for 3 min- (94 ℃ for 40 sec-55 ℃ for 30 sec-72 ℃ for 30 sec) × 35 cycles-72 ℃ for 10 min.

(2) Ligation of purified fragments to cloning vectors

The target DNA molecule is cloned by using pMD19-T simple vector of TaKaRa company, and the ligation reaction system and the procedure are slightly improved by referring to the specification.

Reaction system (5 μ l):

reaction conditions are as follows: 30min at 16 ℃; 4 ℃ overnight.

(3) Transformation of E.coli

1) Thawing freshly prepared or frozen-stored E.coli TOP10 competent cells at-70 ℃ on ice;

2) adding 5 μ l of the ligation product of the purified fragment and the cloning vector into 100 μ l of competent cells, gently mixing, and performing ice bath for about 30 min;

3) heating in 42 deg.C water bath for 90sec, and rapidly placing on ice for 3-5 min;

4) adding 800 μ l LB liquid culture medium, shaking at 37 deg.C and 100rmp for 1 h;

5) centrifuging at 4000rmp for 3min, sucking off 800 μ l of culture medium at the upper layer, and mixing the rest bacteria solution;

6) the bacterial liquid is smeared on an LB screening culture plate containing Amp and is inversely cultured at 37 ℃ for overnight.

(4) Positive clone screening and sequencing analysis

Selecting a single colony from a screening culture plate, inoculating the single colony in an LB liquid culture medium, and shaking the bacteria at 37 ℃ and 250rmp overnight; and directly carrying out PCR detection on the recombinant transformant by taking the overnight cultured bacterial liquid as a template.

Reaction system:

reaction procedure:

the clone with positive PCR detection of the bacterial liquid is sent to Yingjun biotechnology company (Shanghai) for sequencing and identification. The length of the lnc11 sequence is 215bp, and the sequence is shown in SEQ NO. 1.

Example 2 lnc11 overexpression vector construction

An overexpression vector of the lnc11 gene was constructed using Gateway technology. Using specific PCR primers (lnc 11 primer in example 1), PCR amplification was performed using cDNA as a template, and lnc11 was constructed as an entry vector. The entry carrier is pCR ^TM8/GW/TOPO^TMvector (Invitrogen). The reaction system is as follows: 10-20ng of Fresh PCR product (purified); salt solution 1. mu.l; pCR ^TM8/GW/TOPO^TMvector 1. mu.l; add sterile ddH₂O make up to 6. mu.l. The reaction procedure is as follows: standing at room temperature for 30 min.

Selecting positive clones from the screening culture plate to carry out PCR detection and sequencing verification, and carrying out LR reaction on the entry vector carrying the lnc11 gene and the plant expression vector pBI 121. The vector plasmid is shown in FIG. 1. The reaction system is as follows: 100ng of linerizedentry clone; 1.5. mu.l of a normalized destination vector (100 ng/. mu.l); LR clone IIenzyme mix 2. mu.l; TE (pH 8.0) was added to make up to 10. mu.l; reaction conditions are as follows: at 25 ℃ for 1 h. After LR reaction, the Lnc11 gene is introduced into a plant expression vector pBI121, and a strong expression promoter P35S is assembled and assembled at the 5' end of the Lnc11 gene, so that the Lnc11 gene can be efficiently expressed in a poplar body; the strong terminator NOS is assembled at the 3' end of the lnc11 gene, so that the transcription of the lnc11 gene can be effectively terminated; assembling an NPT II gene expression cassette on a vector plasmid, and using the NPT II gene expression cassette as a screening marker of transgenic poplar, wherein the screening of transgenic poplar can be performed by using kanamycin; the LB and RB sequences are assembled in the vector plasmid, and the lnc11 gene expression frame and the screening marker gene NPT II assembled between the sequences are integrated into the chromosome of the poplar receptor cell. The successful construction of the over-expression vector is confirmed through PCR detection and sequencing verification, and is named as 35S: -lnc11, the gene is positioned behind a promoter P35S, and under the drive of the promoter P35S, lnc11 can be efficiently expressed in poplar.

Example 3 construction, transformation and knockout efficiency detection of CRISPR/Cas9 knockout vector of lnc11

3 Target sites, namely Target1, Target2 and Target3, are selected as final Target sites according to the positions of the Target sites in the DNA sequence of lnc11 and the possible off-Target conditions of the Target sites, and the sequence information of the Target sites is shown in Table 1. Further, sgRNA primers were designed based on the sequences of these three target sites (table 2). The selected CRISPR/Cas9 vector contains NPTII resistance gene and has kanamycin resistance, and Cas9 protein is driven by P35S promoter. The 3 sgRNA expression cassette intermediate vectors are pYLsgRNA-AtU3d/LacZ vector driven by Arabidopsis AtU3d promoter, pYLsgRNA-AtU3b vector driven by AtU3b promoter and pYLsgRNA-AtU6-1 vector driven by AtU6-1 promoter respectively. Three Arabidopsis promoters and 3 sgRNAs are firstly connected by using overlapping PCR method to form 1 expression cassette of AtU3d, Target 1-AtU 3b, Target 2-AtU 6-1 and Target 3. Then AtU3d, Target 1-AtU 3b, Target 2-AtU 6-1 and Target3 expression cassettes are connected to a CRISPR/Cas9 vector by a gold-gate cloning method, and finally, a CRISPR/Cas9-lnc11 knockout vector with lnc11 as a Target is successfully constructed. Primers required for vector construction are shown in Table 2. The CRISPR/Cas9 knock-out vector of lnc11 (CRISPR/Cas9-lnc11) was transferred into poplar protoplasts. After 24h dark culture, DNA extraction was performed. Primers were designed at both ends of the target site for PCR amplification and then sequenced. Sequencing analysis revealed that the target site 1 had a 5.6% knock-out efficiency, the target site 2 had a 5.4% knock-out efficiency, and the target site 3 had a higher knock-out efficiency of 71.1% (FIG. 2). The results show that all three target sites successfully carry out gene editing and have higher knockout efficiency, which indicates that the CRISPR/Cas9 gene editing system is feasible in poplar trees.

Table 1 Inc 11 CRISPR/Cas9 target site sequences

TABLE 2 primers required for lnc11 knockout vector construction and detection

Example 4 genetic transformation of lnc11 overexpression vector

The constructed 35S-lnc 11 overexpression vector is transferred into an agrobacterium strain EHA105 by a liquid nitrogen freeze thawing method, and the lnc11 gene is transferred into poplar by agrobacterium mediation. The method comprises the following specific steps:

1) selecting positive single colony, inoculating in 50mL LB liquid culture medium, shaking at 28 deg.C and 250rpm to OD₆₀₀A value of about 0.5;

2) the bacterial liquid is subpackaged in a 50mL centrifuge tube, 1400Xg is centrifuged for 5 minutes, the supernatant is removed, and bacterial plaque is collected;

3) resuspending with an equal volume of MS liquid medium without sucrose;

4) cutting the leaf edge of a leaf of a fresh mountain poplar about one month, putting the cut into a bacterial liquid, and shaking for half an hour at the temperature of 25 ℃ and the speed of 90 rpm;

5) taking out the leaf disc, and transferring the leaf disc into a differentiation culture medium without antibiotic to perform dark culture for 2 days;

6) washing leaf discs with MS liquid culture medium without sugar, and culturing in differentiation culture medium without antibiotic under normal light for 1 week;

7) after 1 week, the leaf discs are put into a differentiation medium added with antibiotics for culture;

8) when the leaf disc grows into buds, transferring the buds into a strong seedling culture medium;

9) finally transferring the grown plantlets into an MS culture medium.

Example 5 detection and phenotypic Observation of transgenic plants

The real-time quantitative PCR technology is utilized to detect the expression condition of the exogenous gene at the RNA level, and the expression level of lnc11 in the over-expression transgenic poplar is found to be remarkably increased (figure 3). Phenotypic observations using one month-old lnc11 overexpressing transgenic poplar revealed that adventitious roots of lnc11 transgenic poplar were significantly reduced (fig. 4). 5 strains of each strain are selected for counting the number of adventitious roots, and the number of the adventitious roots of the lnc11 transgenic poplar is found to be significantly less than CK (figure 5), which indicates that lnc11 is an important lncRNA for regulating and controlling the formation of the adventitious roots of the poplar. The quantity of the adventitious roots of the poplar can be regulated and controlled by over-expressing or knocking out lnc11, and the poplar with different rooting quantity can be cultivated according to requirements, so that the method has important application value in the molecular breeding process of the poplar.

Sequence listing

<110> Yangzhou university

<120> poplar long-chain non-coding RNA lnc11 and application thereof

<160>1

<170>SIPOSequenceListing 1.0

<210>1

<211>215

<212>DNA/RNA

<213>RNA lnc11(RNA lnc11)

<400>1

tcactattta tttcccatca agaaatgacc acttttccat gccagcgttt actcaaccat 60

aaaccaatac catgaaacaa caccatacag cctctacaac attcatttca agattcattt 120

aagctcaagc tcattctttc ttgtaccaaa acacagccta aaacatttat tagttcaaaa 180

accaatacaa aaccaacaac ccagataaaa tctca 215

Claims

1. The nucleotide sequence of the long non-coding RNA lnc11 of the poplar is shown in SEQ NO. 1.

2. An overexpression vector comprising the long non-coding poplar lnc11 of claim 1.

3. The overexpression vector according to claim 2, wherein: the overexpression vector is a strong expression promoter P35S assembled and assembled at the 5' end of lnc 11.

4. The carrier of claim 2, wherein: the overexpression vector is formed by assembling a strong terminator NOS at the 3' end of lnc 11.

5. The carrier of claim 2, wherein: the over-expression vector is assembled with an NPT II gene expression cassette which is used as a screening marker of transgenic poplar.

6. The multi-target CRISPR/Cas9 knock-out vector of lnc11 of claim 1.

7. The multi-target CRISPR/Cas9 knockout vector of lnc11 of claim 6, wherein the CRISPR/Cas9 knockout vector is CRISPR/Cas9-lnc11 vector, contains NPTII resistance gene, has kanamycin resistance, Cas9 protein is driven by P35S promoter; the target site sequence of CRISPR/Cas9 of the lnc11 is TAAACGCTGGCATGGAAAAG; GTTTATGGTTGAGTAAACGC, respectively; TCTTGAAATGAATGTTGTAG are provided.

8. A host cell comprising the long non-coding RNA lnc11 of poplar of claim 1, the overexpression vector of claim 2, or the multi-target CRISPR/Cas9 knockout vector of lnc11 of claim 6.

9. The use of the long non-coding RNA lnc11 of claim 1 for regulating adventitious root formation in poplar.