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.
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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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