阅读说明：本技术 抑制OsHIS1基因表达的植物连锁表达载体及其构建方法和应用 (Plant-linked expression vector for inhibiting OsHIS1 gene expression and construction method and application thereof ) 是由 韶也 赵炳然 袁定阳 彭彦 余东 吕启明 毛毕刚 柏连阳 袁隆平 于 2020-07-01 设计创作，主要内容包括：本发明公开了一种抑制OsHIS1基因表达的植物连锁表达载体,其包括抑制OsHIS1基因表达的元件、育性恢复基因元件、花粉致死基因元件和荧光标记基因元件。还公开了该植物连锁表达载体的构建方法,其得到的植物连锁表达载体转化不育系后,可以通过β三酮类除草剂杀死色选漏检的含有转基因成分的繁殖系,转基因效果精准,除杂效率高。本发明还公开了该植物连锁表达载体在第三代杂交水稻制种或机械化混播混收制种中的应用,可以有效规避第三代杂交水稻制种或机械化混播混收制种使用不育系因色选漏检带来的杂交制种及获得的杂交种子转基因污染风险,操作简单、高效。(The invention discloses a plant-linked expression vector for inhibiting OsHIS1 gene expression, which comprises an element for inhibiting OsHIS1 gene expression, a fertility restorer gene element, a pollen lethal gene element and a fluorescent marker gene element. The obtained plant linked expression vector can kill a breeding line containing transgenic components which is missed in color selection by beta triketone herbicide after being transformed into a sterile line, and has accurate transgenic effect and high impurity removal efficiency. The invention also discloses application of the plant linkage expression vector in third generation hybrid rice seed production or mechanized mixed sowing mixed harvesting seed production, can effectively avoid hybrid seed production and obtained hybrid seed transgenic pollution risk brought by sterile line color selection omission in third generation hybrid rice seed production or mechanized mixed sowing mixed harvesting seed production, and is simple and efficient to operate.)

1. A plant-linked expression vector for inhibiting OsHIS1 gene expression is characterized by comprising an element for inhibiting OsHIS1 gene expression, a fertility restorer gene element, a pollen lethal gene element and a fluorescent marker gene element.

2. The plant-linked expression vector of claim 1, wherein the element that inhibits the expression of OsHIS1 gene comprises a CRISPII silencing element or an RNAi silencing element.

3. The plant linkage expression vector according to claim 2, wherein the CRISPRi silencing element is obtained by linking the CIRSPRi target double strand of the plant herbicide resistance gene to the vector pHdzCas9-KRAB by enzymatic ligation, and the nucleotide sequence of the CIRSPRi target double strand of the plant herbicide resistance gene is shown in SEQ ID No.11 and SEQ ID No. 12.

4. The plant-linked expression vector of claim 2, wherein the RNAi silencing element is obtained by ligating a CDS sequence of a plant herbicide resistance gene, a reverse complement thereof and a Linker sequence into a vector pEGRNAi together by enzymatic ligation, the CDS sequence and the reverse complement thereof being separated by the Linker sequence; the CDS sequence is shown as SEQ ID NO.3, the reverse complementary sequence is shown as SEQ ID NO.4, and the Linker sequence is shown as SEQ ID NO. 5.

5. The plant-linked expression vector of any one of claims 1 to 4, wherein the pollen lethal genetic element comprises the promoter sequence Pg47 shown IN SEQ ID No.7, the cDNA sequence of the ZMAA1 gene shown IN SEQ ID No.1, and the IN2-1 terminator sequence shown IN SEQ ID No. 8; the fluorescent marker gene element comprises a 1tp promoter sequence shown as SEQ ID NO.9, a DsRed gene cDNA sequence shown as SEQ ID NO.2 and a PINII terminator sequence shown as SEQ ID NO. 10; the fertility restorer gene element comprises an EAT1 gene, a PTC1 gene, a TDR gene, a CYP704B2 gene or a PTB1 gene, wherein the nucleotide sequence of the EAT1 gene is shown as SEQ ID No.6, the nucleotide sequence of the PTC1 gene is shown as SEQ ID No.13, the nucleotide sequence of the TDR gene is shown as SEQ ID No.14, the nucleotide sequence of the CYP704B2 gene is shown as SEQ ID No.15, and the nucleotide sequence of the PTB1 gene is shown as SEQ ID No. 16.

6. A method for constructing the plant-linked expression vector according to any one of claims 1 to 5, comprising the steps of: firstly, constructing an element for inhibiting OsHIS1 gene expression, and then connecting the element for inhibiting OsHIS1 gene expression into a plant ternary linked expression vector at least containing a fertility restorer gene element, a pollen lethal gene element and a fluorescent marker gene element through recombination reaction to obtain the plant linked expression vector.

7. The method of constructing according to claim 6, wherein the element that inhibits the expression of OsHIS1 gene comprises CRISPII silencing element or RNAi silencing element;

the construction method of the RNAi silencing element comprises the following steps: firstly, respectively amplifying a CDS sequence, a reverse complementary sequence and a Linker sequence in rice OsHIS1, then connecting amplification products into a vector pEGRNAi together through enzyme digestion ligation reaction, wherein the CDS sequence and the reverse complementary sequence are separated by the Linker sequence, and then obtaining the RNAi silencing element;

the construction method of the CRISPR silencing element comprises the following steps: firstly, mixing two single-stranded nucleotides shown in SEQ ID NO.11 and SEQ ID NO.12 in equal amount, forming a CIRPRII target double-strand with a sticky end after denaturation and annealing, and then connecting the CIRPRII target double-strand into a carrier pHdzCas9-KRAB through enzyme digestion and ligation reaction by using Bsa1 enzyme to obtain the CRISPII silencing element.

8. The method of claim 7, wherein the RNAi silencing element is constructed by using forward and reverse primers for amplifying the CDS sequence as shown in SEQ ID NO.17 and SEQ ID NO.18, respectively, forward and reverse primers for amplifying the reverse complement sequence as shown in SEQ ID NO.19 and SEQ ID NO.20, respectively, and forward and reverse primers for amplifying the Linker sequence as shown in SEQ ID NO.21 and SEQ ID NO.22, respectively.

9. The use of the plant-linked expression vector according to any one of claims 1 to 5 or obtained by the construction method according to any one of claims 6 to 8 in third generation hybrid rice seed production or mechanized mixed sowing and mixed harvest seed production.

10. The application according to claim 9, characterized in that the method of application comprises the steps of:

(1) transforming the plant linkage expression vector into a sterile line of the beta triketone herbicide resistance to obtain a breeding line sensitive to the beta triketone herbicide;

(2) selfing and maturing the breeding line obtained in the step (1), wherein selfing progeny comprises a breeding line containing a transgenic element and a sterile line not containing the transgenic element, screening by a color selector, and separating fluorescent breeding line seeds from non-fluorescent sterile line seeds;

(3) and (3) removing impurities from the sterile line seeds screened by the color sorter in the step (2) by adopting a beta triketone herbicide, removing the breeding line seeds which are missed in color sorting and contain quaternary transgenic elements, and further performing third-generation hybrid rice seed production or mechanical mixed sowing and mixed harvesting seed production on the sterile line seeds obtained after impurity removal.

Technical Field

The invention belongs to the technical field of hybrid rice breeding, and particularly relates to a plant-linked expression vector for inhibiting OsHIS1 gene expression, and a construction method and application thereof.

Background

The development of the rice heterosis utilization technology in China goes through three times. The first generation hybrid rice is three-line hybrid rice with cytoplasmic (nucleoplasm interaction) male sterile line as genetic tool, and the three-line sterile line has stable fertility, but the hybridization matching is limited by restoring and protecting relationship. The second generation hybrid rice is two-line hybrid rice with photo-thermo-sensitive genic male sterile line as a genetic tool, and overcomes the problem of the combination of parents and parents being not free; however, the fertility of the sterile line is influenced by the external light and temperature conditions, so that the risk of seed production failure exists. The third generation hybrid rice uses the common genic male sterile line (i.e. genetic engineering genic male sterile line) as a genetic tool to utilize heterosis, overcomes the technical bottlenecks of the first and second generation technologies, has the advantages of stable fertility and free matching and is the development direction of the hybrid rice technology.

Propagation and sorting of genetic engineering genic male sterile lines are core technologies of third generation hybrid rice, and batch propagation and endosperm fluorescence sorting technologies of genetic engineering sterile line seeds are realized (see patent CN 201210426678.7). In order to screen out a propagation line containing transgenic components in the sterile line color selection process, related patents also introduce double insurance of glume color on the basis of the original endosperm fluorescent marker, and the purity of the sterile line is further improved by two color selections (see patents CN201910699895.5, CN 201910699890.2). However, in the actual color sorting process, because a certain color sorting error exists in the color sorting machine, the method still mixes a reproductive line containing transgenes in the sterile line, which brings the risk of transgenic pollution to the further hybrid seed production of the sterile line.

Disclosure of Invention

The invention aims to solve the technical problem of overcoming the defects and defects in the background technology, and provides a plant linked expression vector for inhibiting OsHIS1 gene expression and a construction method and application thereof, wherein the purity of a third generation hybrid rice sterile line and hybrid seeds thereof can be improved by constructing the plant linked expression vector for inhibiting OsHIS1 gene expression and combining other functional genes and combining a color selection technology and a herbicide.

In order to solve the technical problems, the technical scheme provided by the invention is as follows:

a plant-linked expression vector for inhibiting OsHIS1 gene expression comprises an element for inhibiting OsHIS1 gene expression, a fertility restorer gene element, a pollen lethal gene element and a fluorescent marker gene element.

The above-mentioned linked expression vector, preferably, the element inhibiting the expression of OsHIS1 gene comprises a CRISPR silencing element or an RNAi silencing element. CRISPRi (clustered regularly-spaced short palindromic repeat interference) is co-expressed using inactive dCas9 protein and a customizable single guide rna (sgrna), and conjugation of dCas9 to a transcriptional repressor domain can strongly silence expression of multiple target endogenous genes.

More preferably, the CRISPR silencing element is obtained by connecting the CIRSPR target double strand of the plant herbicide resistance gene into a carrier pHdzCas9-KRAB through enzyme digestion and connection reaction, and the nucleotide sequence of the CIRSPR target double strand of the plant herbicide resistance gene is shown as SEQ ID No.11 and SEQ ID No. 12.

More preferably, the RNAi silencing element is obtained by connecting a CDS sequence of a plant herbicide resistance gene, a reverse complementary sequence of the CDS sequence and a Linker sequence into a vector pEGRNAi together through enzyme digestion ligation reaction, wherein the CDS sequence and the reverse complementary sequence of the CDS sequence are separated by the Linker sequence; the CDS sequence is shown as SEQ ID NO.3, the reverse complementary sequence is shown as SEQID NO.4, and the Linker sequence is shown as SEQ ID NO. 5.

Preferably, the pollen lethal gene element comprises a Pg47 promoter sequence shown as SEQ ID NO.7, a ZMAA1 gene cDNA sequence shown as SEQ ID NO.1 and an IN2-1 terminator sequence shown as SEQ ID NO. 8; the fluorescent marker gene element comprises a 1tp promoter sequence shown as SEQ ID NO.9, a DsRed gene cDNA sequence shown as SEQ ID NO.2 and a PINII terminator sequence shown as SEQ ID NO. 10; the fertility restorer gene element comprises an EAT1 gene (Os04g0599300), a PTC1 gene (Os09g0449000), a TDR gene, a CYP704B2(Os02g0120500) gene (Os03g0168600) or a PTB1 gene (Os05g0145000), wherein the nucleotide sequence of the EAT1 gene is shown as SEQ ID NO.6, the nucleotide sequence of the PTC1 gene is shown as SEQ ID NO.13, the nucleotide sequence of the TDR gene is shown as SEQ ID NO.14, the nucleotide sequence of the CYP704B2 gene is shown as SEQ ID NO.15, and the nucleotide sequence of the PTB1 gene is shown as SEQ ID NO. 16.

Based on a general inventive concept, the present invention provides a method for constructing the plant-linked expression vector, comprising the following steps: firstly, constructing an element for inhibiting OsHIS1 gene expression, and then connecting the element for inhibiting OsHIS1 gene expression into a plant ternary linked expression vector at least containing a fertility restorer gene element, a pollen lethal gene element and a fluorescent marker gene element through recombination reaction to obtain the plant linked expression vector.

In the above construction method, preferably, the element inhibiting the expression of the OsHIS1 gene comprises a CRISPRi silencing element or an RNAi silencing element;

the construction method of the RNAi silencing element comprises the following steps: firstly, respectively amplifying a CDS sequence, a reverse complementary sequence and a Linker sequence in rice OsHIS1, then connecting amplification products into a vector pEGRNAi together through enzyme digestion ligation reaction, wherein the CDS sequence and the reverse complementary sequence are separated by the Linker sequence, and then obtaining the RNAi silencing element;

the construction method of the CRISPR silencing element comprises the following steps: firstly, mixing two single-stranded nucleotides shown in SEQ ID NO.11 and SEQ ID NO.12 in equal amount, forming a CIRPRII target double-strand with a sticky end after denaturation and annealing, and then connecting the CIRPRII target double-strand into a carrier pHdzCas9-KRAB by using Bsa1 enzyme through enzyme digestion and ligation reaction to obtain the CRISPII silencing element.

More preferably, the forward and reverse primers for amplifying the CDS sequence are respectively shown as SEQ ID NO.17 and SEQ ID NO.18, the forward and reverse primers for amplifying the reverse complementary sequence are respectively shown as SEQ ID NO.19 and SEQ ID NO.20, and the forward and reverse primers for amplifying the Linker sequence are respectively shown as SEQ ID NO.21 and SEQ ID NO. 22.

Preferably, the recombination reaction is carried out by inserting 400ng of a ternary linked expression vector comprising a fertility restorer gene, a pollen lethal gene and a fluorescent marker gene into 120ng of an element for suppressing expression of a herbicide resistance gene of a plant, 5 × CE II Buffer 4. mu.l, expase II 2. mu.l and ddH₂O to 20. mu.l, and then reacted at 37 ℃ for 30 min.

Based on a general inventive concept, the invention also provides an application of the plant linkage expression vector in third generation hybrid rice seed production or mechanized mixed sowing and mixed harvesting seed production.

The application preferably comprises the following steps:

(1) transforming the plant linkage expression vector into a sterile line of the beta triketone herbicide resistance to obtain a breeding line sensitive to the beta triketone herbicide;

(2) selfing and maturing the breeding line obtained in the step (1), wherein selfing progeny comprises a breeding line containing a transgenic element and a sterile line not containing the transgenic element, screening by a color selector, and separating fluorescent breeding line seeds from non-fluorescent sterile line seeds;

(3) and (3) removing impurities from the sterile line seeds screened by the color sorter in the step (2) by adopting a beta triketone herbicide, removing the breeding line seeds which are missed in color sorting and contain quaternary transgenic elements, and further performing third-generation hybrid rice seed production or mechanical mixed sowing and mixed harvesting seed production on the sterile line seeds obtained after impurity removal.

Preferably, in the step (1), the plant-linked expression vector is transformed into a sterile line resistant to the beta triketone herbicide through agrobacterium-mediated transformation; in the step (2), the number of the propagation line of the transgenic element contained in the selfed progeny and the number of the sterile line without the transgenic element respectively account for half; in the step (3), the method for removing impurities from the sterile line seeds obtained by screening the color sorter by adopting the beta triketone herbicide specifically comprises the following steps: coating the sterile line seeds with a beta triketone herbicide, adding the beta triketone herbicide in the seed soaking and germination accelerating process of the sterile line seeds or spraying the beta triketone herbicide in the field after the sterile line seeds are sowed and planted.

Preferably, the beta triketone herbicide includes any one or more of Sulcotrione (Sulcotrione), Mesotrione (Mesotrione), benzobicyclon (tembotrione), tembotrione (tembotrione) and furansulcotrione (tefuryltrione). The OsHIS1 gene is an endogenous gene of rice and can endow the rice with resistance to beta-triketone herbicides, and after the function of the gene is deleted, the gene cannot influence the normal growth of the rice, but can make the rice sensitive to the beta-triketone herbicides.

The technical scheme of the invention is based on the following principle: transferring 4 closely linked gene elements of an element for inhibiting OsHIS1 gene expression, a fertility restorer gene element, a pollen lethal gene element and a fluorescent marker gene element into a sterile line to obtain a hybrid single plant of a propagation line carrying transgenes, wherein in the selfing and fructification process of the hybrid single plant carrying the transgenes, pollen grains carrying the transgenes are aborted due to the action of the pollen lethal gene and cannot participate in fertilization, while pollen grains not carrying the transgenes and female gametes (half of each of the female gametes carrying the transgenes and the female gametes not carrying the transgenes) are normally fertilized and fructified to obtain the propagation line carrying the transgenes and the progeny of the sterile line not carrying the transgenes. Because the propagation line carrying the transgene also contains the fluorescence labeling gene, the identification and sorting can be carried out by the color sorting technology, and the propagation line seed emitting fluorescence is preliminarily separated from the sterile line seed not emitting fluorescence. However, due to certain errors in the color selection technology, a reproductive line containing transgenes is still doped in the sterile line after color selection, at the moment, the reproductive line carrying the transgenes contains an element for inhibiting the expression of the OsHIS1 gene, so that the reproductive line is sensitive to the beta-triketone herbicide, and the transgenic reproductive line doped in the sterile line can be efficiently removed by spraying, soaking or coating the beta-triketone herbicide, so that the purities of the sterile line (female and male sterile male parent and female parent) and hybrid seeds thereof are improved.

Compared with the prior art, the invention has the beneficial effects that:

1. the invention develops a plant linkage expression vector, after a plant linkage expression vector transformation sterile line is formed by an element for inhibiting OsHIS1 gene expression, a fertility restorer gene, a pollen lethal gene and a fluorescent marker gene, the element for inhibiting OsHIS1 gene expression can effectively inhibit the expression of rice OsHIS1 gene, so that the rice is sensitive to beta-triketone herbicides, and a breeding line containing transgenic components and subjected to color sorting and omission detection can be killed by the beta-triketone herbicides under the combination of the action of the fertility restorer gene, the pollen lethal gene and the fluorescent marker gene, so that the transgenic effect is accurate, and the impurity removal efficiency is high.

2. The application method of the invention can improve the purity of the sterile line (female and male sterile male and female parents) and the hybrid seeds thereof, is used for the third generation hybrid rice seed production or the mechanized mixed sowing and mixed harvest seed production, and can effectively avoid the transgenic pollution risk of the hybrid seed production caused by color selection omission.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to these drawings without creative efforts.

FIG. 1 is a schematic diagram of the operation of the method for using the plant-linked expression vector of the present invention;

FIG. 2 is a schematic structural view of OsHIS1-RNAi silencing element pEGRNAiPubi-H-OsHIS 1;

FIG. 3 is a schematic structural diagram of OsHIS1-CRISPR silencing element pHdzCas9-KRAB-OsHIS 1;

FIG. 4 is a schematic structural diagram of a plant ternary linked expression vector pDsRed-PTC-ZMAA;

FIG. 5 is a schematic structural diagram of a quaternary linkage expression vector pDsRed-PTC-ZMAA-OsHisRNAi;

FIG. 6 is a schematic structural diagram of a quaternary linkage expression vector pDsRed-PTC-ZMAA-OsHisCRISSPRi;

FIG. 7 is a schematic structural diagram of a plant ternary linked expression vector pDsRed-PTB-ZMAA;

FIG. 8 is a schematic structural diagram of a quaternary linkage expression vector pDsRed-PTB-ZMAA-OsHisRNAi;

FIG. 9 is a schematic structural diagram of a quaternary linkage expression vector pDsRed-PTB-ZMAA-OsHisCRISSPRi.

Detailed Description

In order to facilitate understanding of the invention, the invention will be described more fully and in detail with reference to the accompanying drawings and preferred embodiments, but the scope of the invention is not limited to the specific embodiments below.

Unless otherwise defined, all terms of art used hereinafter have the same meaning as commonly understood by one of ordinary skill in the art. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention.

Unless otherwise specifically stated, various raw materials, reagents, instruments, equipment and the like used in the present invention are commercially available or can be prepared by existing methods.