CN112522300A - Method for cultivating broad-spectrum bacterial leaf streak resistant rice, primer and expression cassette - Google Patents

Abstract

Translated fromChinese

本发明公开了一种培育广谱抗细菌性条斑病水稻的方法及引物和表达盒，该方法包含：通过编辑OsSULTR3；6基因启动子区，使位于OsSULTR3；6基因转录起始位点上游的第422位至第403位的靶标序列产生突变，获得广谱抗细菌性条斑病水稻；其中，所述靶标序列为如SEQ ID No.1所示的核苷酸序列。本发明的方法首次提出通过基因编辑育种构建抗细菌性条斑病水稻的方法，该方法基于CRISPR/Cas9系统编辑OsSULTR3；6基因启动子区，获得的抗病水稻无转基因标记，培育的新品种与自然突变结果相似，可以广泛应用于各种感病水稻的抗性育种，不受水稻品种限制，定向改变水稻特性，周期短，可控性高。

The invention discloses a method, primers and expression cassettes for cultivating broad-spectrum antibacterial leaf stripe rice. The method comprises: editing the OsSULTR3;6 gene promoter region to make the OsSULTR3;6 gene transcription start site upstream The target sequence from position 422 to position 403 of , is mutated to obtain broad-spectrum anti-bacterial leaf spot rice; wherein, the target sequence is the nucleotide sequence shown in SEQ ID No.1. The method of the invention proposes for the first time a method for constructing bacterial leaf spot resistance rice through gene editing and breeding. The method is based on the CRISPR/Cas9 system to edit the OsSULTR3; Similar to the results of natural mutation, it can be widely used in the resistance breeding of various susceptible rice. It is not limited by rice varieties, and can change rice characteristics directionally, with a short cycle and high controllability.

Description

Method for cultivating broad-spectrum bacterial leaf streak resistant rice, primer and expression cassette

Technical Field

The invention relates to a method for cultivating broad-spectrum bacterial leaf streak resistant rice, in particular to a method for cultivating broad-spectrum bacterial leaf streak resistant rice, a primer and an expression cassette.

Background

Bacterial leaf streak of rice caused by Xanthomonas oryzae pv. oryzicola, hereinafter referred to as Xoc, is one of the important bacterial diseases that endanger rice production. The disease is commonly generated in rice areas in the mountains of Qinling and Huaihe south of China, can cause 40 to 60 percent of rice yield reduction when being serious, and is an epidemic disease in China at present. At present, pesticides are mainly used for preventing and treating the bacterial streak disease in agricultural production, but the harm of the bacterial streak disease can only be temporarily relieved by using the pesticides for preventing and treating the bacterial streak disease, and the cost is high, the pollution is heavy and the safety is low. The pathogenic bacteria always have variation in the long-term exposure to the medicament pressure, so that the pesticide effect is reduced. The most economic and effective means for preventing and treating bacterial leaf streak is to breed resistant varieties, but the reports about the bacterial leaf streak resistant rice are few.

With the continuous and intensive research on xanthomonas, it is found that the pathogenic factor of the xanthomonas is mainly caused by a class of important Transcription factor like effector protein (TALE), and the TALE is mainly caused by combining EBE (TALE binding element) in the promoter region of the rice susceptibility gene and inducing the overexpression of the downstream susceptibility gene, so that the rice is attacked. Studies have shown that the pathogenicity of Xoc depends mainly on the induction of OsSULTR3 by Tal2 g; 6 gene over-expression, all Xoc virulence strains published in NCBI contain the DNA sequence of the virulence factor Tal2g in their complete sequence, so that the main virulence factor of these Xoc virulence strains is Tal2 g. The mutation of the EBE sequence of the promoter region of the disease-sensitive gene can cause that TALE protein can not be combined with EBE, thereby reducing the expression level of the disease-sensitive gene when bacteria are infected and improving the disease resistance of rice.

The CRISPR/Cas9(Clustered regulated Short Palindromic Repeats/CRISPR associated proteins 9, hereinafter referred to as CRISPR/Cas9) gene editing system is a gene site-directed editing technique that has been widely used in various species in recent years. The CRISPR/Cas9 system recognizes and binds to a specific gene sequence through a single-stranded guide RNA (sgRNA) and guides Cas9 protein to cut it to break the gene, and finally introduces mutation at the site through a DNA damage repair mechanism of the cell. At present, the technology is not applied to edit rice OsSULTR 3; 6 the report of constructing disease-resistant rice in the gene promoter region.

Disclosure of Invention

The invention aims to provide a method, primers and an expression cassette for cultivating broad-spectrum bacterial streak resistant rice, and OsSULTR3 is edited based on a CRISPR/Cas9 system; 6 gene promoter region to obtain the rice resisting bacterial leaf streak.

In order to achieve the above objects, the present invention provides a method for breeding broad-spectrum bacterial leaf streak resistant rice, comprising: byediting osultr 3; 6 gene promoter region, such that it is located in OsSULTR 3; 6, carrying out mutation on a target sequence from 422 th site to 403 th site upstream of a transcription start site of the gene to obtain broad-spectrum bacterial leaf streak resistant rice; wherein, the target sequence is a nucleotide sequence shown as SEQ ID No. 1; the mutation is G base deletion at the 4 th position in the nucleotide sequence shown in SEQ ID No. 1.

Preferably, the OsSULTR3 is edited by the CRISPR/Cas9 system; 6 gene promoter region, wherein the recombinant vector pYLCRISPR/Cas9-TT2 of the CRISPR/Cas9 system is obtained by converting TT2-sgRNA and pYLCRISPR/Cas9-MT into escherichia coli after enzyme digestion, connection and recombination; the TT2-sgRNA is obtained by performing two PCR reactions by taking a pOsU6a-TT2-gRNA plasmid as a template, and the nucleotide sequence of the TT2-sgRNA is shown in SEQ ID NO. 13; wherein, the pOsU6a-TT2-gRNA plasmid is obtained by connecting a pU6a-gRNA vector and a TT2-gRNA annealing product, the TT2-gRNA annealing product is an annealing product of a primer designed according to the target sequence, and the nucleotide sequences of the primer TT2-gRNA-F, TT2-gRNA-R designed according to the target sequence are respectively shown as SEQ ID NO.2 and SEQ ID NO. 3.

Preferably, in the two PCR reactions, there are two PCR reactions in the first round, and the primers of one PCR reaction are: the nucleotide sequences are respectively shown as a primer U-F, TT2-gRNA-R shown in SEQ ID No.4 and SEQ ID No.3, and the other primer for PCR reaction is as follows: primer TT2-gRNA-F, sgRNA-R with the nucleotide sequence shown as SEQ ID No.2 and SEQ ID No. 5; in the second round of reaction, the products of two PCR reactions in the first round are used as templates, and the primers of the PCR reactions are as follows: the nucleotide sequence is shown as primer B1-F, BL-R shown in SEQ ID No.6 and SEQ ID No. 7.

Preferably, the pU6a-gRNA vector is subjected to enzyme digestion before being connected with the TT2-gRNA annealing product, and the adopted enzyme is BsaI enzyme.

Preferably, the recombinant strain EHA105/pYLCRISPR/Cas9-TT2 of the CRISPR/Cas9 system is obtained by transferring the recombinant vector pYLCRISPR/Cas9-TT2 into agrobacterium EHA 105.

Preferably, the method further comprises: infecting the mature embryo induced callus of the rice with the recombinant strain EHA105/pYLCRISPR/Cas9-TT2, and culturing the plant on a hygromycin antibiotic-containing culture medium to obtain a transgenic positive plant; using the total DNA of the transgenic positive plant as a template, amplifying by using primers pTT2-F, pTT2-R with nucleotide sequences shown as SEQ ID No.8 and SEQ ID No.9 respectively, sequencing the amplified product fragments, judging the editing condition of a target site by analyzing a sequencing peak diagram, and selecting a rice strain with mutation at the target site; extracting total DNA of plant leaves with mutation at target sites as a template, amplifying by primers hyg-F, hyg-R with nucleotide sequences shown as SEQ ID No.10 and SEQ ID No.11 respectively, carrying out gel electrophoresis on the amplified product, and screening a homozygote broad-spectrum disease-resistant rice strain which does not contain a hygromycin label and has edited target sites, so as to obtain the broad-spectrum bacterial leaf streak resistant rice.

Another object of the present invention is to provide OsSULTR3 of rice having a broad spectrum of resistance to bacterial leaf streak; 6 in the promoter region of the gene, OsSULTR 3; 6 gene promoter region as shown in SEQ ID No.1 nucleotide sequence of the 4 th G base deletion.

Another purpose of the invention is to provide a primer for amplifying the total DNA of the transgenic positive plant, and the nucleotide sequence of the primer is shown as SEQ ID No.8 and SEQ ID No. 9.

Another object of the present invention is to provide a gRNA expression cassette, which comprises a TT2-gRNA annealing product in a reaction system, wherein the TT2-gRNA annealing product is obtained by annealing primers having nucleotide sequences shown in SEQ ID No.2 and SEQ ID No.3, respectively, after heating.

Another object of the present invention is to provide the use of said method for constructing rice resistant to bacterial leaf streak.

The method, the primers and the expression cassette for cultivating broad-spectrum bacterial leaf streak resistant rice have the following advantages:

the method of the invention firstly provides a method for constructing the bacterial streak resistant rice by gene editing breeding, and the method edits OsSULTR3 based on a CRISPR/Cas9 system; 6 gene promoter region, in OsSULTR 3; 6 gene transcription initiation site upstream 422 th to 403 th sequence has G base deletion, and through the base deletion of the target site, the rice resisting bacterial leaf streak is obtained.

Drawings

FIG. 1 is OsSULTR 3; 6 gene structure and guide RNA target sequence diagram.

FIG. 2 is OsSULTR3 of homozygous mutant plants; 6 gene target sequence diagram.

FIG. 3 shows the results of gel electrophoresis of PCR screening of gene-edited plants that do not contain the hygromycin gene.

FIG. 4 shows the statistics of lesion spots obtained by inoculating Xoc GX01 with gene-edited rice GT 0105.

Note: in FIG. 2, A is the target sequence; b is a sequencing result; in FIG. 3, M is 100bp plus Marker; 1, taking the total DNA of the cinnamomum japonicum red No.1 as a PCR result of a template; 2 PCR results using GT0105 total DNA as template; 3, the PCR result takes the plasmid pYLCRISPR/Cas9-TT2 as a template; in FIG. 4, wild type rice Guihong No.1 is used as a control; a is a leaf macula map; b is a statistical plot of lesion length, showing a significant difference in lesion length by T-test two-tailed analysis P <0.05 compared to wild-type results.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Experimental example 1 rice OsSULTR3 based on CRISPR/Cas9 system; site-directed mutagenesis of 6 gene promoter region

1. selection of sgRNA (small guide RNA) target sites

Landing on an NCBI website (https:// www.ncbi.nlm.nih.gov /) to obtain rice OsSULTR 3; 6(Gene ID:4324697) Gene sequence of the promoter region of the Gene. According to the design principle of a target site, selecting a target site sequence in a region which can be recognized and induced by the rice bacterial leaf streak pathogen, wherein the selected target site is located in OsSULTR 3; 6 the specific target sequence of the rice containing NGG sequence on the antisense chain of the gene promoter region is a target site sequence TT2(SEQ ID No.1), TT2 is located in OsSULTR 3; 6 from position 422 to position 403 upstream of the transcription start site of the gene (see FIG. 1).

2. Construction of sgRNA expression cassette

(1) Synthesizing a pair of complementary primers TT2-gRNA-F (SEQ ID No.2)/TT2-gRNA-R (SEQ ID No.3) according to the selected target sites;

(2) dissolving the primers TT2-gRNA-F/TT2-gRNA-R into 100 mu M mother solution by using TE buffer solution, adding 1 mu L of each primer into 98 mu L of 0.5x TE buffer solution, mixing and diluting to 1 mu M, carrying out annealing at 90 ℃ for 30s, moving to room temperature, slowly cooling to finish annealing, and obtaining TT2-gRNA annealing products;

(3) mu.g of pU6a-gRNA plasmid was digested with 10U BsaI enzyme (NEB, BsaI-HF, cat # R3733L) for 20min (blunt ends were not digested for a long time) in a 20. mu.L reaction, and the enzyme was inactivated at 70 ℃ for 5 min. The common use of freezing preservation;

(4) connecting a gRNA expression cassette, adding a reaction system according to the following table, reacting at room temperature (20-25 ℃) and connecting for 10-20min to obtain a connection product pOsU6a-TT 2-gRNA.

Table 1 shows the reaction system of gRNA expression cassette

3. Amplification of TT2-sgRNA

(1) First round PCR:

the pOsU6a-TT2-gRNA plasmid is used as a template to carry out two PCR reactions, wherein one PCR reaction is amplified by using a primer U-F (SEQ ID No.4)/TT2-gRNA-R (SEQ ID No.3), the product is A (a fragment containing a target site and a promoter), the other PCR reaction is amplified by using a primer TT2-gRNA-F (SEQ ID No.2)/sgRNA-R (SEQ ID No.5), and the product is B (a fragment containing the target site and the gRNA).

The PCR reaction conditions are as follows: 95 ℃ for 1min, then circulating for 10 times at 95 ℃ for 15s, 55 ℃ for 15s and 68 ℃ for 30s, and finally circulating for 20 times at 95 ℃ for 15s, 60 ℃ for 15s and 68 ℃ for 30 s.

(2) Second round PCR:

mixing products A, B obtained from the first round of PCR reaction in equal proportion and using H₂O is diluted by 20 times, 1 mu L of the mixed solution is taken as a template, and the primer B1-F (SEQ ID No.6)/BL-R (SEQ ID No.7) is used for amplification to obtain an amplification product TT2-sgRNA (SEQ ID No.13) (comprising a U6a promoter, a target site, the sgRNA and a terminator).

The PCR reaction conditions are as follows: 1min at 95 ℃, then circulating for 20 times at 10s at 95 ℃, 10s at 55 ℃ and 10s at 72 ℃ to obtain an amplification product TT 2-sRNA.

(3) The amplification product TT2-sgRNA was purified and recovered by using a DNA purification and recovery kit (Tiangen DNA purification and recovery kit, DP 214).

4. Construction of Cas9-gRNATT2 expression vector containing TT2-sgRNA sequence

(1) Carrying out enzyme digestion on the TT2-sgRNA and the pYLCISPR/Cas 9-MT vector obtained in the previous step by BsaI respectively, wherein the enzyme digestion reaction conditions are that enzyme digestion is carried out at 37 ℃ for 30min and at 73 ℃ for 2min, and two enzyme digestion fragments are connected and recombined, and the connection conditions are that the temperature is 10 ℃ for 5min and the temperature is 20 ℃ for 5 min; and (3) performing 10-15 cycles, and transforming the ligation product into escherichia coli to obtain a recombinant vector pYLCRISPR/Cas9-TT 2.

The vectors pU6a-gRNA and pYLCRISPR/Cas9-MT were derived from the group of Liu dazzling subjects of the university of agriculture of south China, and are disclosed in the documents Ma X, Zhang Q, Zhu Q, et al.A. Robust CRISPR/Cas9 System for Convenient, High-Efficiency Multiplex Genome Editing in monocarding and Dicot Plants [ J ]. molecular plant,2015,8(8): 1274-.

(2) Transferring the recombinant vector pYLCRISPR/Cas9-TT2 obtained in the step (1) into agrobacterium EHA105 in an electric transformation mode to obtain an EHA105/pYLCRISPR/Cas9-TT2 strain;

(3) infecting the callus induced by the mature embryo of the rice variety Guihong No.1 with the EHA 105/pYLCISPR/Cas 9-TT2 strain obtained in the step (2), and taking the successfully regenerated and rooted plant on the culture medium containing hygromycin antibiotics as a transgenic positive plant;

(4) the detection of the editing condition of the target site of the transgenic rice specifically comprises the following steps:

extracting total DNA of regenerated plant leaves as a template, and amplifying OsSULTR3 by using a primer pTT2-F (SEQ ID No.8)/pTT2-R (SEQ ID No.9) as a primer; 6 gene promoter region 515bp segment, sequencing the amplified product segment, and analyzing the sequencing peak image to judge the editing state of the target site. Selecting a rice strain with mutant target sites (the amplified fragment of the mutant strain is 514bp, and the sequence is shown as SEQ ID NO. 12), wherein the specific editing condition is shown as figure 2, andOsSULTR 3; 6 gene promoter region has 1 base G deletion.

The method comprises the steps of extracting total DNA of plant leaves (leaves of homozygous plants obtained by selfing) with mutation at target sites as a template, amplifying by using hyg-F (SEQ ID No.10)/hyg-R (SEQ ID No.11) primers, carrying out gel electrophoresis on amplification products, screening out a homozygous broad-spectrum disease-resistant rice line which does not contain a transgenic label (hygromycin label) and is edited at the target sites, and naming GT0105 as a specific screening condition as shown in FIG. 3, wherein the total DNA of GT0105 and Guihong No.1 as the template and the hyg-F/hyg-R primers cannot amplify a hygromycin gene fragment of about 320bp, and the pYLCRISPR/Cas9-TT2 as the template and the hyg-F/hyg-R primers can amplify a hygromycin gene fragment of about 320bp, which indicates that the transgenic plasmid of GT0105 rice has been removed in the selfing process.

Example 2 detection of disease resistance of disease-resistant Rice against Xoc

The main pathogenic mode of xanthomonas is combination of TAL effector (transcription activation effector) with host EBE and induces expression of downstream susceptible gene to cause rice to be attacked, and the pathogenicity of Xoc mainly depends on the induction of OsSULTR3 by Tal2 g; 6 overexpression of the gene.

The Xoc GX01 strain (the GENEBANK number of NCBI is CP043403.1) is selected as a representative for detecting the disease resistance of the rice edited by a gene. Shaking Xoc GX01 in NB (polypeptone 5g/L, beef extract 3g/L, yeast extract 1g/L, sucrose 10g, optimum pH 7.0) culture medium overnight (28 deg.C, 200rpm), centrifuging the cultured bacterial liquid to obtain thallus, washing with deionized water once, re-suspending with deionized water, adjusting concentration to OD₆₀₀＝0.5。

Inoculating Xoc GX01 strain to exocarpium Cinnamomi Immaturus No.1 and GT0105 by osmolysis method, placing the rice in greenhouse for continuous culture after inoculation, measuring lesion length on 14 days after inoculation and counting, and repeating the experiment for 3 times.

As shown in FIG. 4, the lesion length of the GT0105 strain inoculated with Xoc GX01 is significantly shorter than that of the wild type Guihong No.1 inoculated with the same, indicating that the GT0105 strain has resistance to rice bacterial streak pathogens.

While the present invention has been described in detail with reference to the preferred embodiments, it should be understood that the above description should not be taken as limiting the invention. Various modifications and alterations to this invention will become apparent to those skilled in the art upon reading the foregoing description. Accordingly, the scope of the invention should be determined from the following claims.

Sequence listing

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Claims (10)

1. A method of growing a broad spectrum of rice resistant to bacterial leaf streak, the method comprising:

by editing osultr 3; 6 gene promoter region, such that it is located in OsSULTR 3; 6, carrying out mutation on a target sequence from 422 th site to 403 th site upstream of a transcription start site of the gene to obtain broad-spectrum bacterial leaf streak resistant rice; wherein, the target sequence is a nucleotide sequence shown as SEQ ID No. 1; the mutation is G base deletion at the 4 th position in the nucleotide sequence shown in SEQ ID No. 1.

2. A method of breeding broad spectrum bacterial streak resistant rice plants according to claim 1, wherein the OsSULTR3 is edited by CRISPR/Cas9 system; 6 gene promoter region, wherein the recombinant vector pYLCRISPR/Cas9-TT2 of the CRISPR/Cas9 system is obtained by converting TT2-sgRNA and pYLCRISPR/Cas9-MT into escherichia coli after enzyme digestion, connection and recombination;

the TT2-sgRNA is obtained by performing two PCR reactions by taking a pOsU6a-TT2-gRNA plasmid as a template, and the nucleotide sequence of the TT2-sgRNA is shown in SEQ ID NO. 13; wherein, the pOsU6a-TT2-gRNA plasmid is obtained by connecting a pU6a-gRNA vector and a TT2-gRNA annealing product, the TT2-gRNA annealing product is an annealing product of a primer designed according to the target sequence, and the nucleotide sequences of the primer TT2-gRNA-F, TT2-gRNA-R designed according to the target sequence are respectively shown as SEQ ID NO.2 and SEQ ID NO. 3.

3. A method of growing broad-spectrum rice resistant to bacterial leaf streak according to claim 2, wherein there are two PCR reactions in the first round of said two PCR reactions, and the primers for one PCR reaction are: the nucleotide sequences are respectively shown as a primer U-F, TT2-gRNA-R shown in SEQ ID No.4 and SEQ ID No.3, and the other primer for PCR reaction is as follows: primer TT2-gRNA-F, sgRNA-R with the nucleotide sequence shown as SEQ ID No.2 and SEQ ID No. 5; in the second round of reaction, the products of two PCR reactions in the first round are used as templates, and the primers of the PCR reactions are as follows: the nucleotide sequence is shown as primer B1-F, BL-R shown in SEQ ID No.6 and SEQ ID No. 7.

4. The method for breeding broad-spectrum rice resistant to bacterial leaf streak according to claim 2, wherein the pU6a-gRNA vector is subjected to enzyme digestion before being ligated with the TT2-gRNA annealing product, and the enzyme used is BsaI enzyme.

5. The method for breeding broad spectrum bacterial streak resistant rice plant according to claim 2, wherein the recombinant strain EHA105/pYLCRISPR/Cas9-TT2 of the CRISPR/Cas9 system is obtained by transferring the recombinant vector pYLCRISPR/Cas9-TT2 into agrobacterium EHA 105.

6. A method of growing broad spectrum bacterial leaf streak resistant rice according to claim 5, further comprising:

infecting the mature embryo induced callus of the rice with the recombinant strain EHA105/pYLCRISPR/Cas9-TT2, and culturing the plant on a hygromycin antibiotic-containing culture medium to obtain a transgenic positive plant;

using the total DNA of the transgenic positive plant as a template, amplifying by using primers pTT2-F, pTT2-R with nucleotide sequences shown as SEQ ID No.8 and SEQ ID No.9 respectively, sequencing the amplified product fragments, judging the editing condition of a target site by analyzing a sequencing peak diagram, and selecting a rice strain with mutation at the target site;

extracting total DNA of plant leaves with mutation at target sites as a template, amplifying by primers hyg-F, hyg-R with nucleotide sequences shown as SEQ ID No.10 and SEQ ID No.11 respectively, carrying out gel electrophoresis on the amplified product, and screening a homozygote broad-spectrum disease-resistant rice strain which does not contain a hygromycin label and has edited target sites, so as to obtain the broad-spectrum bacterial leaf streak resistant rice.

7. OsSULTR3 of broad-spectrum rice resistant to bacterial leaf streak; 6 in the promoter region of the gene, characterized by OsSULTR 3; 6 gene promoter region as shown in SEQ ID No.1 nucleotide sequence of the 4 th G base deletion.

8. A primer for amplifying total DNA of transgenic positive plants as claimed in claim 6, wherein the nucleotide sequence of the primer is shown in SEQ ID No.8 and SEQ ID No. 9.

9. A gRNA expression cassette is characterized in that a reaction system of the gRNA expression cassette comprises TT2-gRNA annealing products, and the TT2-gRNA annealing products are obtained by annealing primers with nucleotide sequences shown as SEQ ID No.2 and SEQ ID No.3 respectively after heating.

10. Use of the method according to any one of claims 1 to 6 for the construction of rice resistant to bacterial leaf streak.

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