Method for transforming rape

A rapeseed, transgenic technology, applied in biochemical equipment and methods, horticultural methods, botanical equipment and methods, etc., can solve the problems of low callus induction rate, difficult bud point differentiation, and difficult rooting of transformed seedlings in rapeseed transformation

Active Publication Date: 2017-12-15
BEIJING DABEINONG BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] The purpose of the present invention is to provide a method for transforming rapeseed, which can effectively overcome the technical defects of the prior...

Method used

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  • Method for transforming rape
  • Method for transforming rape
  • Method for transforming rape

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no. 1 example

[0137] The first embodiment, construction of recombinant expression vector and transformation of recombinant expression vector into Agrobacterium

[0138] 1. Construct a recombinant cloning vector containing the target gene

[0139] The EPSPS nucleotide sequence was connected to the cloning vector pGEM-T (Promega, Madison, USA, CAT: A3600), and the operation steps were carried out according to the instructions of the pGEM-T vector produced by Promega Company to obtain the recombinant cloning vector DBN01-T, and its construction process Such as figure 1 Shown (wherein, Amp represents the ampicillin resistance gene; f1 represents the replication origin of phage f1; LacZ is the LacZ initiation codon; SP6 is the SP6 RNA polymerase promoter; T7 is the T7 RNA polymerase promoter; cEPSPS is the 5-ene Alcoholpyruvylshikimate-3-phosphate synthase gene nucleotide sequence (SEQ ID NO: 1); MCS is a multiple cloning site).

[0140] Then, the recombinant cloning vector DBN01-T was transfo...

no. 2 example

[0147] The second embodiment, the acquisition of transgenic rape plants

[0148] 1. Obtaining of explants

[0149] Soak the seeds of Brassica napus variety westar in 70% (v / v) alcohol for 1min, then sterilize with 30% (v / v) sodium hypochlorite solution for 30min, then rinse at least 3 times with sterile water; The washed seeds were inoculated on the germination solid medium (B5 salt 3.1g / L, B5 vitamin, sucrose 20g / L, agar 8g / L, pH5.6), at a temperature of 25±2°C and a photoperiod of 16 / The seedlings were cultivated under the condition of 8h. When the seedling age is 5-7 days, the hypocotyls of the seedlings are cut into sections of about 1.0 cm and used as genetic transformation recipients for future use.

[0150] In the germination solid medium of the present embodiment, B5 salt can also be N6 salt (concentration is 3.95g / L) or MS salt (concentration is 4.3g / L), and the concentration of sucrose can be 5-100g / L Above-mentioned composition all can carry out arbitrarily comb...

no. 3 example

[0181] The third embodiment, using TaqMan to verify the rape plant transferred to the EPSPS nucleotide sequence

[0182] About 100 mg of the leaves of rape plants transferred to the EPSPS nucleotide sequence after the above two rooting treatments were taken as samples, and the genomic DNA was extracted with Qiagen's DNeasy Plant Maxi Kit, and the expression of the EPSPS gene was detected by the Taqman probe fluorescence quantitative PCR method. copy number. At the same time, wild-type rape plants were used as a control, and detection and analysis were carried out according to the above method. The experiment was repeated 3 times, and the average value was taken.

[0183] The specific method for detecting the copy number of the EPSPS gene is as follows:

[0184] Step 11, take respectively 100 mg of leaves of rape plants and wild-type rape plants transferred to EPSPS nucleotide sequences after the above two kinds of rooting treatments, and grind them into homogenate with liquid ...

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Abstract

The invention relates to a method for transforming rape. The method comprises the following steps: obtaining hypocotyls or segments thereof after rape seeds germinate; infecting the hypocotyls or segments thereof by using agrobacterium strains; performing callus induction culturing on the infected hypocotyls or segments thereof; screening and culturing rape calluses by using a selecting agent, and performing bud differentiation culturing on screened rape resistance calluses on a ZT-containing differentiation culturing medium; culturing young rape seedlings growing from the rape buds on a first rooting culture medium which contains cytokinin and auxin and a second rooting culture medium which does not contain any hormone. The method for transforming the rape is high in callus induction rate, high in bud differentiation rate and high in plant survival rate, and facilitates rooting; the transforming efficiency is up to 8 percent.

Description

technical field [0001] The invention relates to a method for plant transformation, in particular to a method for transformation and cultivation of rapeseed. Background technique [0002] Rapeseed is a Brassica plant in the family Brassicaceae, which is divided into three cultivars: Brassica rapa L., Brassica Juncea L. and Brassica napu L., among which Brassica napu L. is grown globally The area is the most extensive, followed by the mustard type, and the cabbage type is the least. At present, rapeseed, as one of the four major oil crops, is the fourth largest genetically modified crop in the world. In 2010, the global planting area of ​​genetically modified rapeseed accounted for 23% of the total global rapeseed area, reaching 7 million hectares 2 , and increased year by year. Since Ooms et al. first used Agrobacterium-mediated method to obtain transgenic rapeseed in 1985, the transgenic research of rapeseed has made breakthroughs in many aspects such as disease resistance...

Claims

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Application Information

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IPC IPC(8): C12N5/04C12N15/82A01H5/00A01H4/00
CPCA01H4/001A01H4/008C12N5/0025C12N15/8205
Inventor 刘雁华李运亭贾志伟宋庆芳
Owner BEIJING DABEINONG BIOTECHNOLOGY CO LTD
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