Identification of capsicum annuum CMS male sterility gene Ca06g18730 based on fertility differential protein and application

A male sterility gene, differential protein technology, applied in application, genetic engineering, plant genetic improvement, etc., can solve problems such as utilization restrictions

Active Publication Date: 2020-10-02
HANGZHOU ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although genetic male sterility is simple, relatively stable, and easy to transfer, it is necessary to pull out the fertile plants in the du...

Method used

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  • Identification of capsicum annuum CMS male sterility gene Ca06g18730 based on fertility differential protein and application
  • Identification of capsicum annuum CMS male sterility gene Ca06g18730 based on fertility differential protein and application
  • Identification of capsicum annuum CMS male sterility gene Ca06g18730 based on fertility differential protein and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1: Observation of anther microstructure of CMS traits of pepper

[0033] Pepper sterile line DH-01-1-1A and maintainer line DH-01-1-1B were sown at the base of Hangzhou Academy of Agricultural Sciences in early November, and transplanted into greenhouses in early March of the following year until early April. The pepper material enters the full blooming period, observe that the anthers of the sterile line DH-01-1-1A are thin and shriveled, no pollen is scattered, and the pistil development is normal, see figure 1 .

[0034] From 8:00 to 9:00 in the morning, the buds are picked in 6 different periods from small to large. Then, use tweezers to peel off the anthers in the buds for later use. Five different periods of pepper sterile line anthers were selected from small to large to make paraffin sections, and the differences in their development process were observed under the Leica DM1000 microscope. The results showed that the anthers of the sterile plants started f...

Embodiment 2

[0035] Example 2: Screening of CMS trait-related proteins in pepper based on quantitative proteomics

[0036] Take the sterile and maintainer anthers of the tetrad stage and the microspore stage, and screen the proteins related to CMS traits by quantitative proteomics technology, which specifically includes the following steps:

[0037] 1) Total protein extraction and enzymatic hydrolysis: Take 2g of anther samples from the sterile line and maintainer line and grind them into powder under liquid nitrogen. Add 4 times the volume of the powder lysis buffer (8 M urea, 1% TritonX- 100, 10 mM dithiothreitol and 1% protease inhibitor), ultrasonic lysis. Centrifuge at 20000 g for 10 min at 4°C, take the supernatant and add trichloroacetic acid with a final concentration of 20%, and let stand at 4°C for 2h. Centrifuge at 12000 g for 3 min at 4°C, discard the supernatant, and wash the pellet three times with pre-cooled acetone. Finally, the precipitate was reconstituted with 8M urea, and ...

Embodiment 3

[0045] Example 3: Genes encoding proteins related to CMS traits in pepper Ca06g18730 Cloning and functional verification

[0046] Combined with bioinformatics tools, the CDS sequence of the corresponding coding gene is obtained according to the screening results of the differential protein, and the fertility-related gene is cloned by inverse PCR technology Ca06g18730 Gene and verify its function in pepper anther development through VIGS technology, including the following steps:

[0047] 1) Ca06g18730 Gene cloning:

[0048] The CDS sequence of CA06g18730 encoding gene was obtained according to the screening results of differential proteins, and the full-length ORF of CA06g18730 gene was cloned using reverse PCR technology.

[0049] 2) CA06g18730 Construction of gene virus silencing vector:

[0050] A pair of specific primers were designed according to the sequence of CA06g18730 gene homology region in the maintainer. The primer sequence is as follows:

[0051] EcoRI CA06g18730-F 5'-CCG...

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Abstract

The invention discloses a capsicum annuum CMS male sterility gene Ca06g18730 based on fertility differential protein. The nucleotide sequence of the capsicum annuum CMS male sterility gene Ca06g18730is shown as SEQ ID NO. Anther specific gene Ca06g18730 related to the capsicum annuum CMS character is identified by utilizing a quantitative proteomics technology, and it is clear that anther of sterile line plants is changed from the meiosis quadruple period, so that pollen abortion is caused; the expression of the maintainer line capsicum annuum Ca06g18730 gene is silenced through a VIGS technology, it is indicated that the CA06g18730 gene plays a key role in the pollen development process, and down-regulation of the CA06g18730 gene is an important factor causing pollen abortion of the CMSsterile line; a VIGS technology is utilized to silence the expression of the capsicum annuum Ca06g18730 gene, so that plant pollen abortion is caused, a capsicum annuum male sterile material is created, and a new material is provided for capsicum annuum breeding.

Description

Technical field [0001] The invention belongs to the technical field of pepper breeding, and relates to specific genes of pepper anther fertility differential protein Ca06g18730 And its application. Background technique [0002] Plant male sterility (male sterility) refers to the phenomenon that plant anthers cannot produce normal and functional pollen, which usually includes the inability of anthers to produce pollen, pollen abortion, and the inability of anthers to crack (Kaul 1988). Because the stamens of male sterile plants cannot produce functional pollen, there is no need to remove the stamens during the hybridization process, saving a lot of manpower, material and financial resources, and since self-pollination of the plants can be avoided, it provides a strong guarantee for the seed purity of the hybrid generation. It provides an effective way for the wide application of heterosis in agricultural production. Capsicum (Capsicumannuum L.) belongs to the Solanaceae (Solanace...

Claims

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Application Information

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IPC IPC(8): C12N15/56C12N15/84C12N1/21A01H5/02A01H6/82
CPCC12N9/244C12N15/8218C12N15/8289C12Y302/01006
Inventor 柴伟国裘劼人忻雅肖文斐王淑珍方献平
Owner HANGZHOU ACAD OF AGRI SCI
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