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sgRNA composition for knocking out anemone vitifolia Buch GhTSTs gene and application of sgRNA composition to creation of anemone vitifolia Buch non-short-fiber mutant

A technology without short follicles and composition, applied in DNA/RNA fragmentation, application, genetic engineering, etc., can solve the problems of few related gene reports, weak target, long time consumption, etc.

Active Publication Date: 2021-05-18
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Cotton lint-free or short-lint mutants are generally obtained through breeding and screening, but this method requires a lot of work, takes a long time, and is not very targeted.
Although transgenic technology is a fast and effective method for cotton genetic improvement, many key genes controlling long fiber development have been identified from cotton, and hormone regulation pathways controlling cotton fiber cell elongation have also been discovered. There are few reports on genes related to the lint-free trait, so the development of a functional gene related to the lint-free trait in cotton is of great significance for the creation of cotton lint-free mutants

Method used

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  • sgRNA composition for knocking out anemone vitifolia Buch GhTSTs gene and application of sgRNA composition to creation of anemone vitifolia Buch non-short-fiber mutant
  • sgRNA composition for knocking out anemone vitifolia Buch GhTSTs gene and application of sgRNA composition to creation of anemone vitifolia Buch non-short-fiber mutant
  • sgRNA composition for knocking out anemone vitifolia Buch GhTSTs gene and application of sgRNA composition to creation of anemone vitifolia Buch non-short-fiber mutant

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0084] Construction of GhTSTs gene CRISPR vector

[0085] (1) Construction of CRISPR knockout vectors pRGEB32-GhTST12 and pRGEB32-GhTST13

[0086] There are a total of 10 GhTSTs genes in cotton. The present invention creates two CRISPR / Cas9 vectors for multiple TST gene knockouts, respectively named pRGEB32-GhTST12 and pRGEB32-GhTST13, referring to the genome sequence and the CRISPR-P website http: / / cbi.hzau.edu.cn / crispr / Search for targets, where pRGEB32-GhTST12 contains gRNA1 and gRNA2, which can knock out 8 GhTSTs genes at the same time; pRGEB32-GhTST13 contains gRNA1 and gRNA3, which can knock out 6 GhTSTs genes at the same time, see Table 3 for details.

[0087] Table 3 Three sgRNA target gene information

[0088]

[0089]Primers were designed according to the 3 sgRNA sequences for two PCR amplifications, and the primer sequences are shown in Table 1.

[0090] Table 1 Information of each amplification primer

[0091]

[0092]

[0093] Use primers pGREB32-7F ...

Embodiment 2

[0099] Genetic transformation of pRGEB32-GhTST12 / GhTST13 vector in cotton

[0100] (1) Cotton genetic transformation mediated by Agrobacterium

[0101] The test material is the upland cotton strain (YZ1). The plump and consistent YZ1 seeds were selected, the seed coat was peeled off, and sterilized with 0.1% mercuric chloride solution for 10-12 minutes. on the surface of MS medium. After 1 day of dark culture at 30°C, seedlings were supported, and dark culture was continued for 4 to 5 days.

[0102] The EHA105 strain in Example 2 was inoculated into 2ml of LB liquid containing 100mg / L spectinomycin and LB liquid containing 100mg / L kanamycin, cultured with shaking at 28°C for 1 day, and the activated bacterial liquid was respectively connected to 20ul in In 20ml of fresh liquid LB containing 100mg / L spectinomycin and fresh liquid LB containing 100mg / L kanamycin, incubate overnight at 28°C with shaking, draw 1ml of turbid bacterial liquid into a 2ml sterile centrifuge tube, an...

Embodiment 3

[0113] Molecular identification of pRGEB32-GhTST12 / GhTST13 transgenic plants

[0114] (1) Positive detection of transgenic plants

[0115] The genomic DNA of the young leaves of the transgenic plants was extracted using the plant genomic DNA extraction kit of Tiangen Biochemical (Beijing) Technology Co., Ltd. 2a or U6-7S and InfTSTs-3a were performed to detect whether there is corresponding T-DNA insertion by PCR respectively. PCR reaction conditions: 94°C pre-denaturation for 5 minutes; 94°C for 30 sec, 58°C for 30 sec, 72°C for 1 min, 28 cycles; 72°C for 5 min.

[0116] image 3 The electropherogram of the positive PCR detection results of the pRGEB32-GhTST12 transgenic material provided by the present invention; lane M represents Marker, WT represents the wild-type receptor material, the corresponding band can be amplified by PCR of the transgenic material, and there is no band in the wild type. The PCR amplification primers for positive detection of mutant materials wer...

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Abstract

The invention provides an sgRNA composition for knocking out anemone vitifolia Buch GhTSTs gene and an application of the sgRNA composition to creation of a anemone vitifolia Buch short-fiber-free mutant, and belongs to the technical field of plant genetic engineering. According to the invention, three sgRNAs for gene editing are designed for conserved fragments of 8 GhTSTs genes, two sets of CRISPR-Cas9 vectors are constructed, and agrobacterium tumefaciens-mediated genetic transformation is performed, so that the anemone vitifolia Buch short fiber-free mutant is obtained. Therefore, the invention provides the application of the GhTSTs gene to creation of the anemone vitifolia Buch non-linter mutant. Besidse, the invention also provides an application of a carrier of which the GhTSTs gene is knocked out to creaction of the anemone vitifolia Buch short-fiber-free mutant. According to the invention, the GhTSTs gene is determined as a target gene for creating the anemone vitifolia Buch non-short-fiber character, the anemone vitifolia Buch non-short-fiber mutant is successfully created, and a material basis is provided for subsequent research on short-fiber generation and non-short-fiber related breeding and screening.

Description

technical field [0001] The invention belongs to the technical field of plant genetic engineering, and in particular relates to an sgRNA composition for knocking out cotton GhTSTs gene and its application in creating cotton linterless mutants. Background technique [0002] Cotton (Anemone vitifolia Buch) is an important economic crop and occupies an important position in the world and our national economy. Cotton fiber is an indispensable raw material for the production of cotton products in the textile industry. Cotton textiles involve all aspects of people's lives, including cotton textiles for clothing, decorative cotton textiles, and industrial cotton textiles. In cotton production, fiber accounts for about 40% and the rest is cottonseed. [0003] Cotton fiber is the most widely used fiber material. Cotton fiber is a seed fiber formed by elongating and thickening the epidermal cells of fertilized ovules, and can be divided into long-staple and short-staple. Its main co...

Claims

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Application Information

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IPC IPC(8): C12N15/29C12N15/113C12N15/84A01H5/10A01H6/60
CPCC07K14/415C12N15/113C12N15/8218C12N15/8261C12N2310/20
Inventor 杨细燕夏林杰邓晋武孙伟男涂礼莉张献龙
Owner HUAZHONG AGRI UNIV
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