Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Application of rape BnLAC2 gene in improvement of cold-resistant early blossoming

A rapeseed and gene technology, applied in the application, genetic engineering, plant genetic improvement and other directions, can solve the problems of cold resistance gene mining and molecular mechanism research, lack of molecular markers and other problems

Active Publication Date: 2022-03-08
INST OF OIL CROPS RES CHINESE ACAD OF AGRI SCI
View PDF4 Cites 1 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Rapeseed is a cool-loving crop, but there are relatively few researches on cold resistance gene mining and molecular mechanism, lack of key molecular markers, and the improvement of cold resistance in rapeseed also urgently needs rich genetic resources

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Application of rape BnLAC2 gene in improvement of cold-resistant early blossoming
  • Application of rape BnLAC2 gene in improvement of cold-resistant early blossoming
  • Application of rape BnLAC2 gene in improvement of cold-resistant early blossoming

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Example 1 Design of sgRNA of Brassica napus BnLAC2 gene CRISPR-Cas9 and construction of carrier BnLAC2-Cas9

[0022] (1) sgRNA sequence determination

[0023] Brassica napus is a tetraploid crop, and the BnLAC2 gene has one copy in each of the two sets of genomes. The nucleotide sequence of the cDNA of the BnLAC2 gene is shown in SEQ ID NO.1. Sequence alignment of the two copies was performed to find the PAM (proto ad ja cent motif) motif (NGG) in the conserved region. The present invention designs two sgRNAs, the sequences of which are shown in SEQ ID NO.2 and SEQ ID NO.3, and both are located in the first exon, and the target sites are as follows figure 1 shown.

[0024] (2) Synthesis of Oligo DNA single strand

[0025] UP oligo: 5'-ATTG+sgRNA sequence-3'

[0026] LOW oligo: 5'-AAAC+sgRNA reverse complementary sequence-3'

[0027] The two pairs of Oligo DNA single-strand sequences of the present invention are shown in SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6, SEQ...

Embodiment 2

[0058] Example 2: Transformation of Agrobacterium GV3101 with BnLAC2-Cas9 vector

[0059] Add 1 μl of BnLAC2-Cas9 to 100 μl of GV3101 Agrobacterium competent cells, mix well, and then ice-bath for 5 minutes, freeze in liquid nitrogen for 1 minute, and bathe in 37°C water for 5 minutes, add 500μl liquid LB medium, 28°C, 200rpm shaker recovery for 1 hour. Take 100 μl of bacterial liquid and spread it on the LB solid culture dish containing 50mg / L kanamycin, 50mg / L gentamycin and 50mg / L rifampicin; culture at 28°C for 2 days, pick a single clone, and inoculate in The LB liquid medium containing 50mg / L kanamycin, 50mg / L gentamycin and 50mg / L rifampicin was cultured overnight at 28°C on a 200rpm shaker, and then carried out PCR identification with the primers on the carrier. Then the Agrobacterium liquid was mixed with 50% glycerol and stored in a -80°C ultra-low temperature refrigerator.

Embodiment 3

[0060] Example 3: BnLAC2-Cas9 Agrobacterium transformation of Brassica napus hypocotyl

[0061] 1. Sowing

[0062] Soak the rapeseed seeds with an appropriate amount of 75% alcohol for 1 minute, pour off the alcohol and wash it with sterile water, pour off the water, and then sterilize with 50% 84 disinfectant (sterile water: commercial 84 solution = 1:1) for 10 minutes. Minutes, after disinfection, pour the disinfectant into the waste liquid tank. For heavily polluted seeds, the sterilization time can be extended to 20 minutes. The seeds were then washed 5 times with an appropriate amount of sterile water. Sow the treated seeds into the M0 medium with sterile tweezers, sow 20-25 seeds per dish, then put the petri dish into a sterile culture box, and cultivate it at 24°C for 5-6 days in the dark.

[0063] 2. Activation and preparation of Agrobacterium

[0064] (1) The day before dipping, antibiotics were added to 100 mL of sterilized liquid LB medium, Agrobacterium strains ...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention discloses application of an oilseed rape BnLAC2 gene in improving cold-resistant early flowering of oilseed rape, sgRNA specifically targeting the oilseed rape BnLAC2 gene is designed, the sgRNA is prepared into an oligo dimer, the oligo dimer and a Cas9 skeleton construct a gene editing carrier, the gene editing carrier is introduced into cabbage type oilseed rape hypocotyl callus through an agrobacterium tumefaciens-mediated genetic transformation technology and regenerated into seedlings, and the cold-resistant early flowering of the oilseed rape is improved. Cas9 nuclease edits a BnLAC2 gene on a genome A under the guidance of sgRNA to generate insertion mutation, phenotypic identification finds that the biomass of the oilseed rape subjected to cold injury treatment of a homozygous mutant strain is improved, and the oilseed rape blooms two weeks ahead of time.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to the application of rapeseed BnLAC2 gene in improving cold resistance and early flowering. Background technique [0002] Rapeseed is the second largest oilseed crop in China, after soybeans. It shoulders the important task of vegetable oil supply in my country, and the rapeseed oil produced accounts for more than 57% of the oil produced by domestic oil crops. At present, China's oil crop self-sufficiency rate is less than 40%, and a large amount of rapeseed still needs to be imported from abroad every year. Therefore, the development of rapeseed not only has an important impact on China's agricultural production and national economy, but also has great significance for the security of national oil supply. [0003] The area of ​​rapeseed in my country's winter rapeseed area accounts for more than 90% of the total area of ​​rapeseed in the country, and it is mainly planted ...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/29C07K14/415C12N15/82A01H5/02A01H6/20
CPCC07K14/415C12N15/827C12N15/8273C12N15/8216
Inventor 吕艳穆罕默德·阿扎尔·侯赛因罗丹程勇邹锡玲
Owner INST OF OIL CROPS RES CHINESE ACAD OF AGRI SCI
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com