阅读说明：本技术 一种基于育性差异蛋白的辣椒CMS雄性不育基因Ca06g18730鉴定及应用 (Identification and application of CMS male sterility gene Ca06g18730 of pepper based on fertility differential protein ) 是由 柴伟国 裘劼人 忻雅 肖文斐 王淑珍 方献平 于 2020-07-09 设计创作，主要内容包括：本发明公开了一种基于育性差异蛋白的辣椒CMS雄性不育基因Ca06g18730,其核苷酸序列如SEQ ID NO.1所示,本发明利用定量蛋白质组学技术鉴定到一种辣椒CMS性状相关的花药特异基因Ca06g18730,明确了不育系植株的花药从减数分裂四分体时期开始变异,造成花粉败育；通过VIGS技术沉默保持系辣椒Ca06g18730基因的表达,说明CA06g18730基因在花粉发育过程中起着关键作用,且CA06g18730基因下调为导致CMS不育系花粉败育的一个重要因素；利用VIGS技术沉默辣椒Ca06g18730基因表达,造成植株花粉败育,创制辣椒雄性不育材料,为辣椒育种提供新的材料。(The invention discloses a fertility differential protein-based pepper CMS male sterility gene Ca06g18730, the nucleotide sequence of which is shown in SEQ ID NO.1, the invention identifies a pepper CMS character-related anther specific gene Ca06g18730 by utilizing a quantitative proteomics technology, and confirms that the anther of a sterile line plant begins to mutate from a meiosis tetrad period to cause pollen abortion; the expression of the conservation line pepper Ca06g18730 gene is silenced by VIGS technology, which shows that the CA06g18730 gene plays a key role in the pollen development process, and the CA06g18730 gene is down-regulated to be an important factor causing CMS sterile line pollen abortion; the VIGS technology is utilized to silence the gene expression of the pepper Ca06g18730, so that the pollen of the plant is aborted, the pepper male sterile material is created, and a new material is provided for pepper breeding.)

1. A hot pepper CMS male sterility gene Ca06g18730 based on fertility differential protein has a nucleotide sequence shown in SEQ ID No. 1.

2. A vector and a recombinant bacterium containing the CMS male sterility gene Ca06g18730 of pepper as claimed in claim 1.

3. The use of the CMS male sterility gene Ca06g18730 of pepper as claimed in claim 1 for regulating the fertility of pepper anther.

4. Use according to claim 3, characterized in that it comprises the following steps:

1) constructing a carrier containing pepper anther fertility related gene Ca06g 18730;

2) the constructed vector of the pepper anther fertility related gene Ca06g18730 is instantaneously transformed into a maintainer line pepper;

3) screening to obtain a transgenic pepper strain with pollen abortion.

5. The use according to claim 4, wherein the vector is pTRV2-CA06g 18730.

Technical Field

The invention belongs to the technical field of pepper breeding, and relates to a specific gene of pepper anther fertility differential proteinCa06g18730And applications thereof.

Background

Plant male sterility (male sterility) refers to the phenomenon of failure of plant anthers to produce normal functioning pollen, and generally includes failure of anthers to produce pollen, pollen abortion, and failure of anthers to split (Kaul 1988). Because the stamens of the male sterile plants can not produce functional pollen, the stamens do not need to be removed in the hybridization process, a large amount of manpower, material resources and financial resources are saved, and because the self-pollination of the plants can be avoided, the powerful guarantee is provided for ensuring the purity of the first filial generation of seeds, and an effective way is provided for the wide application of heterosis (heterosis) in agricultural production. The Capsicum (Capsicum annuum L.) belongs to the Solanaceae (Solanaceae) Capsicum (Capsicum L.) crop, is the second vegetable crop in China, and is second to Chinese cabbage only. The pepper is a normal cross-pollinated vegetable crop, and the heterosis of the F1 generation is obvious. In 1956, the phenomenon of male sterility of hot pepper was first discovered by Martin in sweet pepper, and then the phenomenon of nuclear-cytoplasmic interaction male sterility of hot pepper was discovered (Peterson et al, 1958), and the hot pepper male sterile line has been widely used in hybrid seed production at present. The pepper male sterility used for hybrid seed production can be divided into nuclear male sterility (GMS) which is mainly derived from local variety segregation progeny, X-ray, gamma-ray and EMS-treated generations, and Cytoplasmic Male Sterility (CMS) which is mainly derived from natural mutation, mutagenized progeny and interspecific hybridization generations. Although the nuclear male sterility has simple heredity, stability and easy transformation, the fertile plants in the dual-purpose line need to be pulled out during the seed production, so that the utilization of the nuclear male sterility is limited to a certain extent, and a part of domestic units use the method to produce the seeds. The cytoplasmic male sterility three-line is matched to produce the pepper hybrid seeds, so that the labor cost of manual emasculation, isolation and the like can be saved, the seed purity is improved, and the protection of intellectual property rights and the like are facilitated, so that agricultural experts in various countries gradually pay attention to the research and utilization of the pepper CMS.

Pollen abortion is the phenotype embodiment of the occurrence of plant male sterility, and the whole process and molecular mechanism of pollen development are the basis and key for researching plant male sterility. In recent years, research on development of stamens and pollen and regulation and control of fertility gene expression by using male sterile lines have become hot research in the field of plant breeding. At present, only a few genes related to the development of pepper pollen are reported, so that more genes related to the development of pepper pollen are still needed to be researched and developed so as to create a pepper male sterile material and provide a new material for pepper breeding. Proteomics technology, as a research hotspot in life science today, has become an important way to decipher gene function and contribute to breeding programs in biology. The adoption of proteomics technology to screen protein, a direct product of gene expression affecting plant traits, is one of the more direct and effective methods for mining differential expression functional genes.

Disclosure of Invention

Aiming at the problems in the prior art, the invention aims to provide identification and application of a male sterility gene Ca06g18730 of a pepper CMS based on fertility differential protein.

The invention is realized by the following technical scheme:

the nucleotide sequence of the male sterility gene Ca06g18730 of the pepper CMS based on the fertility differential protein is shown in SEQ ID NO. 1.

A vector and a recombinant bacterium containing the CMS male sterility gene Ca06g18730 of pepper as claimed in claim 1.

The application of the CMS male sterility gene Ca06g18730 of capsicum in regulating and controlling the fertility of capsicum anther.

The application is characterized in that the application of the CMS male sterility gene Ca06g18730 of the pepper in regulating and controlling the pepper anther fertility comprises the following steps:

1) constructing a carrier containing pepper anther fertility related gene Ca06g 18730;

2) the constructed vector of the pepper anther fertility related gene Ca06g18730 is instantaneously transformed into a maintainer line pepper;

3) screening to obtain a transgenic pepper strain with pollen abortion.

The application is characterized in that the vector is pTRV2-CA06g 18730.

The invention is realized by the following experiments:

the cause and the period of the sterility are determined by observing the anther microstructure of CMS plants of the pepper.

The invention relates to a pepper CMS character related protein screening method based on quantitative proteomics. The callose enzyme protein CA06g18730 with almost unchanged content in the sterile line is screened out, and the callose enzyme protein is obviously up-regulated in the pepper maintainer line in the tetrad stage and the microspore stage.

The invention relates to cloning and functional verification of CMS character related protein coding gene Ca06g18730 of pepper. According to the CDS sequence of the gene coding the differential protein CA06g18730, the full-length cloning of the ORF of the CA06g18730 gene is carried out by using the reverse PCR technology. Designing a pair of specific primers according to a CA06g18730 gene homologous region sequence, constructing a CA06g18730 gene virus silencing vector and transforming agrobacterium, infecting pepper plant leaves by injection, observing the development condition of flower organs, and determining the gene function.

The silent pepper plant CA06g18730 gene can create a pepper male sterile material, and provide a new material for pepper breeding.

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

1) the invention utilizes a quantitative proteomics technology to identify anther specific gene Ca06g18730 related to CMS character of pepper. By observing the anther microstructure of the CMS plant of the pepper, the variation of the anther of the sterile line plant from the meiotic tetrad period is determined, the excessive vacuolation of tapetum cells gradually increases radially, so that the tetrad is extruded to the center of the medicine chamber and the protoplast is thick, and the callose around the tetrad can not disintegrate and then gradually disintegrates to cause pollen abortion. Screening anther proteins of CMS sterile line and maintainer line of pepper at different periods by adopting a quantitative proteomics method, and screening callose enzyme protein CA06g18730 with almost unchanged content in the sterile line, wherein the anther proteins are obviously up-regulated in the maintenance line of pepper at tetrad period and microspore period;

2) the total length of the capsicum Ca06g18730 gene is 2146bp, and the nucleotide sequence is shown as SEQID NO. 1;

3) according to the invention, the function of the CA06g18730 gene in pollen development is identified by analyzing the pollen viability of a silent plant through the expression of the Ca06g18730 gene of a silent maintainer line pepper by a VIGS technology, and the finding shows that the CA06g18730 gene silent plant can only produce a small amount of abnormal pollen compared with a control, and the pollen can not germinate. The key role of the CA06g18730 gene in the pollen development process is shown, and the CA06g18730 gene is down-regulated to be an important factor causing CMS sterile line pollen abortion;

4) the VIGS technology is utilized to silence the gene expression of the pepper Ca06g18730, so that the pollen of the plant is aborted, the pepper male sterile material is created, and a new material is provided for pepper breeding.

Drawings

FIG. 1 shows the flower morphology of the sterile line and the maintainer line of pepper, which clearly shows abortion characteristics;

FIG. 2 is an electron microscope image of paraffin sections of anthers of a sterile line and a maintainer line of pepper, and the cause and the occurrence period of pollen abortion are observed;

FIG. 3 is a process flow for constructing pTRV2-CA06g18730 vector;

FIG. 4 shows that pTRV2-CA06g18730 virus vector EcoRI, BamHI double enzyme digestion tests to check whether the virus silencing vector is constructed successfully;

FIG. 5 shows the growth of VIGS-injected albino pepper plants;

FIG. 6 shows comparison of expression levels of the gene of VIGS-silenced plants and the gene of the maintainer line CA06g 18730;

FIG. 7 shows the flower organ morphology of VIGS plants and control plants, and the pollen abortion effect was examined.

Detailed Description

The invention is described in further detail below with reference to the accompanying drawings, and specific embodiments are given.