Method for preparing citrus homozygous mutant by CRISPR/Cas9 mediated gene editing technology

A mutant, citrus technology, applied in the field of genetic engineering, can solve the problem of low efficiency of citrus homozygous mutants, and achieve the effect of improving the efficiency of gene editing

Active Publication Date: 2021-08-06
SOUTHWEST UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the efficiency of CRISPR / Cas9 to obtain homozygous mutants in citrus is low

Method used

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  • Method for preparing citrus homozygous mutant by CRISPR/Cas9 mediated gene editing technology
  • Method for preparing citrus homozygous mutant by CRISPR/Cas9 mediated gene editing technology

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] (1) Construction of p35SCas9 / CsLOB1sgRNA vector

[0021] Taking CsLOB1 (Cs7g27640) in the citrus Wanjincheng genome as the target gene, CRISPR-P 2.0 ( http: / / crispr.hzau.edu.cn / CRISPR2 / ) software in CsLOB1 A 20-nucleotide sgRNA sequence 5'-TCCTGTTTACGGCTGCGCCG-3' was designed on the exon of the gene; a GATTG sequence was added to the 5' end of the sgRNA sequence to obtain a 5'-GATTGTCCTGTTTACGGCTGCGCCG-3' sequence; AAAC sequence is added to the 5' end, and a single base C is added to the 3' end to obtain the 5'-AAACCGGCGCAGCCGTAAACAGGAC-3' sequence;

[0022] The above sequence was synthesized by Beijing Liuhe Huada Gene Technology Co., Ltd. After the sequence was synthesized, it was dissolved to a concentration of 10 μM, and the two sequences were annealed to form double-stranded DNA; Slowly anneal for 2h under the condition of -20℃ for later use.

[0023] The pUC119-gRNA plasmid was digested with BbsI. The digestion reaction system is: BbsI 1μL, Buffer 2μL, pUC119-...

Embodiment 2

[0044] GUS histochemical staining of adventitious buds

[0045] Adventitious buds germinated on the wounds at both ends of the epicotyl stem section were stained with GUS (containing the following components: 100mMNaH 2 PO 4 , 100mM Na 2 HPO 4 , 0.5mM K 4 [Fe(CN) 6 ], 0.5 mM K 3 [Fe(CN) 6 ], 10 mM EDTA-Na 2 , 1 mM X-gluc, 0.1% Sodium azide, 0.1% Triton-100) for GUS histochemical staining.

[0046] The staining results showed that the GUS staining was blue, that is, the GUS-positive buds.

Embodiment 3

[0048] Micro-grafting of GUS-positive buds and secondary grafting in the field

[0049] When the stem of the GUS-positive bud is about 0.5 cm long, it is cut horizontally and slightly grafted on the citrange rootstock;

[0050] After the grafting interface is fully healed, cut the GUS-positive buds horizontally from the base of the citrange rootstock, graft them on the field citrange rootstock according to the method of field cutting, keep them moist with plastic bags for 2 weeks, and remove the plastic bags after they survive .

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Abstract

The invention belongs to the technical field of gene engineering, and particularly relates to a method for preparing a citrus homozygous mutant by a CRISPR / Cas9 mediated gene editing technology. The method comprises the following steps of: co-culturing a citrus epicotyl stem and agrobacterium, immediately performing heat treatment on the epicotyl stem, and then transferring the epicotyl stem into an MS culture medium added with 1mg / L BA, 0.5mg / L IAA and 40mg / L Km for induction. The method can improve the gene editing efficiency of the homozygous mutant.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a method for preparing citrus homozygous mutants based on CRISPR / Cas9-mediated gene editing technology. Background technique [0002] Citrus, also known as citrus, is a plant of the genus Citrus in the family Rutaceae, including many varieties such as mandarin orange, orange, orange, pomelo, and lemon. Citrus fruits are rich in nutrition, containing flavonoids, limonoids, alkaloids, coumarins and vitamins and other active ingredients, which have anti-cancer and cardiovascular disease prevention effects ("Research progress of alkaloids in citrus" , Zhao Xuemei, etc., Chinese Pharmacy, 2008, Volume 19, No. 9, Page 703, Paragraph 1, Lines 1-3, published on May 29, 2008; "Research on the Quality of Flavonoids in Rutaceae Citrus", Shi Rui et al., Chinese Journal of Traditional Chinese Medicine, Vol. 30, No. 6, 2012, page 585, left column, paragraph 1, line 1-3...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82C12N15/113A01H5/00A01H6/78
CPCC12N15/8218C12N15/113C12N2310/20
Inventor 彭爱红邹修平何永睿陈善春许兰珍雷天刚
Owner SOUTHWEST UNIV
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