Method and application of knocking out BnMAX1 gene in Brassica napus by CRISPR-Cas9 system

A Brassica napus and gene technology, applied in the field of genetic engineering, can solve problems such as improving the difficulty of CRISPR-Cas9 targeting, and achieve the effects of increasing yield traits, efficient breeding methods, and simplifying construction steps

Active Publication Date: 2019-01-25
INST OF OIL CROPS RES CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] Although CRISPR technology is a powerful tool for gene function research, it can also be used for gene editing to improve crops. However, in polyploid crops such as wheat and rapeseed, since genes have copies in each subgenome, this greatly improves the ability of CRISPR-Cas9. The difficulty of shooting, only by silencing these homologous copies is a successful shooting

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  • Method and application of knocking out BnMAX1 gene in Brassica napus by CRISPR-Cas9 system
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  • Method and application of knocking out BnMAX1 gene in Brassica napus by CRISPR-Cas9 system

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experiment example 1

[0037] Experimental example 1: sgRNA design of BnMAX1 gene CRISPR-Cas9 in Brassica napus and construction of vectors BnMAX1-Cas9-1 and BnMAX1-Cas9-2

[0038] 1. sgRNA sequence determination

[0039]Brassica napus is a tetraploid crop with two genomes, A and C, and the BnMAX1 gene has one copy in each of the two genomes. Sequence comparison of the two copies was performed to find the PAM (proto adjacent motif) motif (NGG) in the conserved region, and a sequence of 20 bp at the 5' end of the PAM position was the sgRNA sequence. The present invention designs two sgRNAs, the sequences of which are shown in SEQ ID NO.1 and SEQ ID NO.2, and both are located in the first exon, and the target sites are as follows figure 1 , 2 shown.

[0040] 2. Synthesis of Oligo DNA single strand

[0041] The CRISPR-Cas9 vector construction kit used in the present invention was purchased from Hangzhou Baige Biotechnology Co., Ltd. (Cat#BGK01). Therefore, when designing the Oligo DNA single strand...

experiment example 2

[0059] Experimental Example 2: Transformation of Agrobacterium GV3101 with BnMAX1-Cas9-1 and BnMAX1-Cas9-2 Vectors

[0060] Add 5 μl of BnMAX1-Cas9-1 and BnMAX1-Cas9-2 DNA to 100 μl of GV3101 Agrobacterium competent cells, mix well, ice-bath for 5 minutes, freeze in liquid nitrogen for 1 minute, bathe in 37°C water for 5 minutes, add 700 μl of liquid LB culture medium, 28°C, 200rpm shaker recovery culture for 4 hours. After the cultivation, take an appropriate amount of bacterial liquid and spread it on the LB solid plate containing 50mg / L kanamycin, 50mg / L gentamycin and 50mg / L rifampicin; , inoculated in LB liquid medium containing kanamycin, gentamycin and rifampicin, cultured overnight at 28°C at 200 rpm, and then used cas9-F primers and corresponding Low oligo for PCR identification.

[0061] The correctly identified BnMAX1-Cas9-1 and BnMAX1-Cas9-2 Agrobacterium liquids were mixed with 50% glycerol and stored at -80°C.

experiment example 3

[0062] Experimental example 3: BnMAX1-Cas9-1 and BnMAX1-Cas9-2 Agrobacterium transformed brassica napus hypocotyls respectively

[0063] 1. Explant Preparation

[0064] Seed surface disinfection of Brassica napus 862 (spring rape strain collected in the laboratory) and winter rape cultivar Zhongshuang 6 as materials

[0065] (1) Add the seeds into a clean 50 ml centrifuge tube, soak the seeds with 75% alcohol for 5 minutes;

[0066] (2) Pour off the alcohol, add 10ml of 1.5% mercury liter, soak the seeds for 15 minutes, and shake them every few minutes to make the liquid fully contact with the seeds;

[0067] (3) pour off the mercuric acid, and clean the seeds about 4 times with sterilized single distilled water;

[0068] (4) Blot the remaining water with a pipette gun, put the seeds into the M0 medium with tweezers that have been burnt and sterilized in advance, 50 seeds per bottle, and 6 bottles of seeds for each transformation;

[0069] (5) 24 degrees, dark culture for 5...

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Abstract

The invention discloses a method and application of knocking out a BnMAX1 gene in Brassica napus by a CRISPR-Cas9 system, two sgRNAs of specifically targeting Brassica napus BnMAX1 gene are designed and synthesized into oligo dimer, are connected with Cas9 vector, and introduced into hypocotyl callus of Brassica napus by Agrobacterium tumefaciens-mediated transformation to regerated into shoots. Cas9 nuclease was guided by sgRNA to cleave target sequence. Each sgRNA could pass through CRISPR-Cas9 system mediates the cleavage of BnMAX1 gene in A and C genomes, and achieves the goal of gene knockout. Phenotypic identification showed that homozygous mutant lines increased the number of branches and pods per plant, decreased plant height and increased yield.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and specifically relates to two sgRNAs specifically targeting the BnMAX1 gene of Brassica napus and a method for knocking out the BnMAX1 gene of Brassica napus using a CRISPR-Cas9 system, which can be used to cultivate lodging-resistant and high-yield rapeseed varieties. Background technique [0002] Brassica napus L. belongs to the Brassica genus of Brassicaceae. It is the second largest oil crop in the world and one of the most important oil crops in my country. my country's rapeseed planting area and output both rank first in the world, but the yield per unit area is lower than that of Canada, Australia and other countries. The agronomic traits that affect the yield of rapeseed mainly include plant height, effective branch number, branch position and flowering time. The number of branches is one of the important plant type traits related to crop yield. In rapeseed, increasing the n...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/82A01H5/00A01H6/20
CPCC07K14/415C12N15/113C12N15/8213C12N15/8261C12N2310/10C12N2310/20
Inventor 王汉中华玮郑明杨红丽张亮
Owner INST OF OIL CROPS RES CHINESE ACAD OF AGRI SCI
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