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Method for obtaining high-oleic-acid cotton by applying gene editing technology

A gene editing, high oleic acid technology, applied in the application, genetic engineering, recombinant DNA technology and other directions, can solve the problems of insufficient genetic stability, incomplete gene silencing, and the oleic acid content of cotton has not yet been seen, so as to reduce linoleic acid. content, the effect of increasing the content of oleic acid

Active Publication Date: 2019-11-22
SHANDONG COTTON RES CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The RNAi method sometimes has problems such as incomplete gene silencing, easily weakened silencing effect in offspring, and insufficient genetic stability.
However, there is no report on the use of CRISPR-Cas9 technology to increase the content of oleic acid in cotton

Method used

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  • Method for obtaining high-oleic-acid cotton by applying gene editing technology
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  • Method for obtaining high-oleic-acid cotton by applying gene editing technology

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1: Construction of CRISPR / Cas9 gene editing vector

[0026] 1. gRNA target selection

[0027] Taking the cotton FAD2 gene (Gh_A13G1850 and Gh_D13G2238) as the target gene, using the online software CRISPR-Pv2.0 (http: / / crispr.hzau.edu.cn / CRISPR2 / ) to screen the target, select the target sites sgRNA1 and sgRNA2. The cotton FAD2 gene contains two conserved domains, namely DUF3474 (PF11960) and FA_desaturase (PF00487). The target sgRNA1 is located in the DUF3474 domain, and its PAM sequence is CCA, while the target sgRNA2 is located in the FA_desaturase domain, and its PAM sequence is CCG ;

[0028] The sgRNA1 sequence is: 5'-CCATTCCGCCCCACTGTTTTCGC-3';

[0029] The sgRNA2 sequence is: 5'-CCGTCACCACTCGAACACCGGTT-3'.

[0030]2. Construction of dual-target CRISPR / Cas9 gene editing vector

[0031] Linker primers for sgRNA1 and sgRNA2: According to the designed target sites, synthetically construct vector-related primers, see Table 1:

[0032] Table 1 pRGEB32-GhU6...

Embodiment 2

[0038] Example 2: Obtaining of Gene Edited Cotton Plants

[0039] 1. Preparation of sterile receptor material

[0040] The seeds of the upland cotton variety Huamian No. 1 (HM1) were used as materials, and the seed shells were peeled off after sulfuric acid delinting, sterilized with 70% ethanol solution for 1 minute, and then soaked in 0.1% mercuric chloride (HgCl). 2 ) sterilized for 15 minutes, rinsed with sterile water for 5 times, inoculated on the seed germination medium, and cultured in the dark at 22-28°C for 3-5 days before use.

[0041] The seed germination medium is: 1 / 2MS medium macronutrient + 15g / L glucose + 2.5g / L Phytagel, pH value is 5.8-6.0.

[0042] 2. Cultivation of donor Agrobacterium GV3101

[0043] Agrobacterium tumefaciens GV3101 carrying dual-target sgRNA1 and sgRNA2 was inoculated on LB solid medium plates and cultured in the dark at 28°C for 36-48 hours. Use a sterile toothpick to pick a single colony with good growth, inoculate it into LB liquid ...

Embodiment 3

[0056] Embodiment 3: PCR detection of transgenic cotton plants

[0057] 1. DNA Extraction of Transgenic Cotton Plants

[0058] Using Tiangen Biochemical Technology (Beijing) Co., Ltd. to extract T. 0 DNA from transgenic cotton leaves. Specifically include the following steps:

[0059] (1) Cut 80-100 mg of fresh young leaves of each transgenic cotton plant and wild-type cotton (HM1), and immediately add liquid nitrogen to fully grind, and transfer the ground powder to a numbered 1.5ml centrifuge tube;

[0060] (2) Quickly add 400 μl buffer solution GPS and 10 μl RNase A (10 mg / ml), vortex quickly and mix well, then place the centrifuge tube in a 65°C water bath for 15 minutes, invert the centrifuge tube 5 times during the water bath to better mix the samples ;

[0061] (3) Add 100μl buffer GPA, vortex for 1min, centrifuge at 12000rpm for 5min, transfer the supernatant to the filter column CS (the filter column CS is placed in the collection tube), then centrifuge at 12000rp...

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Abstract

The invention discloses a method for obtaining high-oleic-acid cotton by applying a gene editing technology. According to the method, firstly, gRNA targets including sgRNA1 and sgRNA2 are selected, then a pRGEB32-GhU6.9-NPTII carrier is used as a basis, and the fused sgRNA1 and sgRNA2 are inserted into a BasI locus of the pRGEB32-GhU6.9-NPTII carrier; the established carrier is converted into competence escherichia coli, a culture medium slab with kanamycin is coated with the competence escherichia coli for screening, a single colony is selected for culture, PCR identification is conducted, amonoclone with correct sgRNA1 and sgRNA2 sequences is cultured, plasmid is extracted, an agrobacterium-mediated mode is adopted, the plasmid is converted into cotton, a transgenic cotton plant with FAD2 gene mutation is obtained through screening, the oleic acid content of transgenic cotton seeds is obviously increased, and the linoleic acid content of the transgenic cotton seeds is obviously lowered.

Description

technical field [0001] The invention relates to a method for obtaining high oleic acid cotton by applying gene editing technology, and belongs to the technical field of plant genetic engineering. Background technique [0002] Cotton is the world's most important natural fiber crop, as well as an important source of edible oil and protein. The oil content in cottonseed is 15-40%. Cottonseed oil is the fifth largest edible vegetable oil in the world, second only to soybean, palm, rapeseed and sunflower. It is also the main edible vegetable oil consumed by residents in the main cotton producing areas of my country. The fatty acid composition of cottonseed mainly includes palmitic acid (C16:0), stearic acid (C18:0), oleic acid (C18:1), linoleic acid (C18:2) and linolenic acid (C18:3). Compared with other vegetable oils, its palmitic acid content is higher, which makes it have better stability and shortening, and can prolong the shelf life of fried foods; the content of linoleic...

Claims

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Application Information

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IPC IPC(8): C12N15/82C12N9/22A01H5/00A01H6/60
CPCC12N15/8213C12N9/22C12N15/8247Y02P60/87
Inventor 柳展基陈义珍傅明川李浩王立国刘任重
Owner SHANDONG COTTON RES CENT
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