Recombinant expression vector for cultivating cucumber male sterile line and construction method and application thereof
阅读说明：本技术 一种用于培育黄瓜雄性不育系的重组表达载体及其构建方法和应用 (Recombinant expression vector for cultivating cucumber male sterile line and construction method and application thereof ) 是由 杨丽 马明茹 别之龙 于 2020-06-03 设计创作，主要内容包括：本发明公开了一种重组表达载体,为在载体质粒的多克隆位点间插入黄瓜CsMLO12和CsMLO13基因的两个sgRNA靶点,所述黄瓜CsMLO12基因具有如SEQ ID NO:1所示的核苷酸序列,所述黄瓜CsMLO13基因具有如SEQ ID NO:2所示的核苷酸序列,本发明还公开了重组表达载体的构建方法及其在培育黄瓜雄性不育系中的应用,通过构建与花粉管萌发相关基因CsMLO12、CsMLO13的CRISPR/Cas9表达载体,并通过农杆菌介导遗传转化黄瓜,获得Csmlo12、Csmlo13双突变体,发现双突变体的花粉不能正常萌发出花粉管从而使其雄性不育。(The invention discloses a recombinant expression vector, which is characterized in that two sgRNA targets of cucumber CsMLO12 and CsMLO13 genes are inserted between multiple cloning sites of a vector plasmid, the cucumber CsMLO12 gene has a nucleotide sequence shown as SEQ ID NO:1, and the cucumber CsMLO13 gene has a nucleotide sequence shown as SEQ ID NO: 2.)
1. A recombinant expression vector characterized by: the recombinant expression vector is obtained by inserting two sgRNA targets of cucumber CsMLO12 and CsMLO13 genes between multiple cloning sites of a vector plasmid, wherein the cucumber CsMLO12 gene has a nucleotide sequence shown as SEQ ID NO. 1, and the gene codes a protein with an amino acid sequence shown as SEQ ID NO. 3; the cucumber CsMLO13 gene has a nucleotide sequence shown as SEQ ID NO. 2, and the gene codes a protein with an amino acid sequence shown as SEQ ID NO. 4.
2. The recombinant expression vector of claim 1, wherein: the vector plasmid is pKSE402 plasmid.
3. The recombinant expression vector of claim 1, wherein: the nucleotide sequences of the two sgRNA targets are respectively as follows:
CsMLO 12: the sequence shown as SEQ ID NO. 5;
CsMLO 13: the sequence shown in SEQ ID NO. 6.
4. A method of constructing a recombinant expression vector according to any one of claims 1 to 3, comprising the steps of:
s1: designing two sgRNA targets according to cucumber CsMLO12 and CsMLO13 genes, wherein the sgRNA targets are both located in an exon structure domain, and designing and synthesizing primers required for constructing a recombinant expression vector according to sequences of the sgRNA targets;
s2: and performing PCR amplification on the two sgRNAs by using the primers, purifying and recovering an amplification product, and performing enzyme digestion connection, so that the two sgRNA targets are inserted between multiple cloning sites of vector plasmids and a recombinant expression vector is constructed.
5. The method for constructing a recombinant expression vector according to claim 4, wherein the nucleotide sequences of the primers are:
MLO12-DT 1-BsF: the sequence shown as SEQ ID NO. 7;
MLO12-DT 1-F0: the sequence shown as SEQ ID NO. 8;
MLO13-DT 2-R0: a sequence shown as SEQ ID NO. 9;
MLO13-DT 2-BsR: 10, SEQ ID NO.
6. An engineered bacterium comprising the recombinant expression vector of any one of claims 1 to 3.
7. A transgenic Agrobacterium tumefaciens containing the recombinant expression vector of any one of claims 1-3.
8. Use of the recombinant expression vector of any one of claims 1 to 3, the engineered bacterium of claim 6, the transgenic agrobacterium of claim 7 for breeding male sterile lines of cucumber.
9. The use of claim 8, wherein: a CRISPR/Cas9 gene editing system is adopted to knock out CsMLO12 and CsMLO13 gene structural domains of cucumber at fixed points, so that the functions of the corresponding proteins of the genes are deleted, and the pollen tube of the cucumber is caused to germinate defectively, thereby being male sterile.
Technical Field
The invention belongs to the field of plant genetic engineering, and particularly relates to a recombinant expression vector for cultivating a cucumber male sterile line, and a construction method and application of the recombinant expression vector.
Background
Cucumber (Cucumis sativus) is one of the most important vegetable crops in China. At present, the hybrid seed production of the cucumber still needs manual removal of male flowers and bagging pollination, the process is complex and the cost is high. Male sterility in plants is mainly characterized by the inability of the stamens to produce normal functioning pollen, while the pistils develop normally and are able to accept pollen fertilization for fruiting. The male sterility has important application value in the aspect of utilizing the heterosis of the plants, and is an effective way for improving the hybrid seed production efficiency. In the key project of 'key technology integration of main vegetable crop male sterility breeding and fine breed industrialization' of the technical support plan of the fifteen countries of the ministry of science and technology of China, cucumber is one of the important key projects of attack and customs. With the development of bioengineering, it has become possible to create male sterile materials by gene editing methods. The gene editing technology can modify gene sequences on the DNA level, so that the accurate improvement of target characters is realized without the problems of linkage drag of conventional backcross breeding genes and the like. The male sterile genes are found in many crops, but the male sterile genes reported in the cucumber are few, and the application of hybrid seed production in the cucumber is greatly limited.
The MLO gene is originally found in barley, and the mutation of the MLO gene can make barley generate lasting, broad-spectrum and efficient powdery mildew resistance. mlo recessive mutation-mediated powdery mildew resistance is subsequently widely used in breeding against various other crops (Kusch and Panstruga, 2017). The plant MLO gene can be divided into seven evolutionary branches, and has multiple functional effects. Wherein the member of branch V participates in mediating powdery mildew resistance, the member of branch I participates in the regulation of plant root system morphology, and the MLO protein on branch III may be a key regulator of gametophyte function in the fertilization process of angiosperm. As arabidopsis thaliana branch III member AtMLO7 is involved in pollen tube and helper cell recognition (Kessler et al, 2010), the rice branch III member OsMLO12 gene is essential for pollen hydration after pollen grains contact the affinity column heads (Yi et al, 2014); however, the function of the MLO III branch member in melon crops is not clear. The male sterility of plants is mainly reflected in pollen abortion, and we find that the cucumber pollen tube can not normally germinate by editing the CsMLO12 and CsMLO13 genes of the cucumber III branch members, so that the male sterility is caused.
Disclosure of Invention
The first object of the present invention is to provide a recombinant expression vector.
The recombinant expression vector is obtained by inserting two sgRNA targets of cucumber CsMLO12 and CsMLO13 genes between multiple cloning sites of a vector plasmid.
The cucumber CsMLO12 and CsMLO13 genes are derived from any one of genes in cucurbitaceae, Cucumis and cucumber (Cucumis sativusL.), and specifically can be the following genes 1) -2):
1) the coding sequence is the 1 st to 1635 th positions of the 5 'end of CsMLO12 of SEQ ID NO. 1 and the 1 st to 1683 th positions of the 5' end of CsMLO13 of SEQ ID NO. 2;
2) DNA molecule which has more than 90 percent of homology with the gene of 1) and codes protein related to pollen tube germination.
The following proteins of 1) or 2):
1) a protein consisting of amino acid sequences shown by SEQ ID NO. 3 and SEQ ID NO. 4; CsMLO12 of
2) Protein which is derived from the amino acid sequence of SEQ ID NO. 3 or SEQ ID NO. 4 by substitution and/or deletion, and/or addition of one or more amino acid residues and is related to pollen tube germination and is derived from the protein of 1).
The protein can be artificially synthesized, or can be obtained by synthesizing the coding gene and then carrying out biological expression. The genes encoding CsMLO12 and CsMLO13 in 2) above can be obtained by deleting one or several codons of amino acid residues from the DNA sequence shown by 1 to 1635 th bases from the 5 'end of CsMLO12 of SEQ ID NO. 1 and 1 to 1683 th bases from the 5' end of CsMLO13 of SEQ ID NO. 2, and/or performing missense mutation of one or several base pairs.
The protein related to pollen tube germination and the coding gene thereof also belong to the protection scope of the invention.
Preferably, the vector plasmid is pKSE402 plasmid.
Preferably, the nucleotide sequences of the two sgRNA targets are:
CsMLO 12: the sequence shown as SEQ ID NO. 5;
CsMLO 13: the sequence shown in SEQ ID NO. 6.
The second purpose of the invention is to provide a construction method of the recombinant expression vector, which comprises the following steps:
s1: designing two sgRNA targets according to cucumber CsMLO12 and CsMLO13 genes, wherein the sgRNA targets are both located in an exon structure domain, and designing and synthesizing primers required for constructing a recombinant expression vector according to sequences of the sgRNA targets;
s2: and performing PCR amplification on the two sgRNAs by using the primers, purifying and recovering an amplification product, and performing enzyme digestion connection, so that the two sgRNA targets are inserted between multiple cloning sites of vector plasmids and a recombinant expression vector is constructed.
Preferably, the nucleotide sequences of the primers are:
MLO12-DT 1-BsF: the sequence shown as SEQ ID NO. 7;
MLO12-DT 1-F0: the sequence shown as SEQ ID NO. 8;
MLO13-DT 2-R0: a sequence shown as SEQ ID NO. 9;
MLO13-DT 2-BsR: 10, SEQ ID NO.
The third purpose of the invention is to provide an engineering bacterium containing the recombinant expression vector, and the engineering bacterium is recombinant escherichia coli containing the expression vector.
The fourth purpose of the invention is to provide a transgenic agrobacterium containing the recombinant expression vector, wherein the agrobacterium is agrobacterium tumefaciens EHA 105.
The fifth purpose of the invention is to provide the application of the recombinant expression vector, the engineering bacteria and the transgenic agrobacterium in the cultivation of the cucumber male sterile line. The application comprises the steps of knocking out CsMLO12 and CsMLO13 gene structural domains of cucumber cotyledons at fixed points by adopting a CRISPR/Cas9 gene editing system, so that the functions of proteins corresponding to the genes are deleted, and the germination defect of a cucumber pollen tube is caused, thereby enabling the cucumber pollen tube to be male sterile.
The invention constructs CRISPR/Cas9 expression vectors of CsMLO12 and CsMLO13 genes related to pollen tube germination, introduces the genetic transformation into cucumber germplasm, successfully obtains transgenic T0 generations after stable genetic transformation, and separates T1 generation double mutants by selfing and reserving seeds. It was found that pollen tubes could not germinate normally in the transgenic plant T1 generation Csmlo12, Csmlo13 double mutant and that almost no normal seeds could be obtained when the double mutant served as male parent. The experimental results show that the proteins encoded by CsMLO12 and CsMLO13 play an important role in pollen tube germination. The intensive research on the expression and functions of the CsMLO12 and CsMLO13 genes is helpful for analyzing the mechanism of cucumber pollen tube germination, and provides a theoretical basis for cucumber male sterility.
Drawings
FIG. 1 shows a set of CsMLO12 and CsMLO13 gene sgRNA expression elements.
Fig. 2 shows the target site editing situation of transgenic cucumber T1 generation.
FIG. 3 shows pollen tube germination in WT and Csmlo12/13 double mutant medium. ac and bd are pollen of WT and Csmlo12/13 double mutants, respectively, cd is pollen tube germination after culturing pollen of WT and Csmlo12/13 double mutants on a culture medium for 5.5 h.
FIG. 4 shows the statistics of the number of normal seeds of single melon when Csmlo12/13 double mutants were used as parents for crossing.
Detailed Description
The present invention will be further described with reference to the following specific examples.