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Cotton plant event a-6, as well as detection primer and detection method thereof

A technology of cotton and primer pairs, applied in botany equipment and methods, biochemical equipment and methods, angiosperms/flowering plants, etc., can solve the problem of inability to obtain the molecular characteristics of flanking sequences of gene integration of transformation events, inconvenient aggregation of transgenic traits, Uncertainty of integrated copy number and integrated position of Bt cotton

Active Publication Date: 2013-03-27
BIOCENTURY TRANSGENE CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In the early studies of Bt cotton in my country, due to the limitation of transformation methods, the obtained Bt cotton had uncertain integration copy number and integration position, and it was not easy to grasp the transgenic traits in breeding, and the Bt cotton selected by different breeders Insect resistance of insect cotton varieties is quite different
At the same time, because the molecular characteristics of the flanking sequence of the gene integration of the transformation event cannot be obtained, it is not convenient for further aggregation of new transgenic traits, which limits its further application

Method used

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  • Cotton plant event a-6, as well as detection primer and detection method thereof
  • Cotton plant event a-6, as well as detection primer and detection method thereof
  • Cotton plant event a-6, as well as detection primer and detection method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1. Plant expression vector construction

[0030]The 35S promoter containing double enhancers was amplified from the vector pCambia2300, with Xba I and BamH I at both ends. The primer sequences are shown in SEQ ID No: 1 and SEQ ID No: 2. The PCR product was cloned into pMD after enzyme digestion In -18T, the pMD-35S recombinant vector was obtained. In order to increase the recombination rate of the expression cassette, the SSR sequence TMB0066 with a large number of topoisomerase II recognition sites from the cotton gene was cloned, and a plant expression vector with TMB0066 as the MAR sequence was constructed to increase the integration efficiency of the inserted sequence. The sequence of TMB0066 is shown as SEQ ID No:3. Hind III and Xba I sites were introduced into its two ends respectively, and cloned into pMD-35S to obtain the recombinant vector pMD-TMB0066-35S.

[0031] The plasmid containing the OK (Omega&Kozak)-Bt-PS (Processing&Splicing sequence) fragme...

Embodiment 2

[0032] Example 2. Genetic transformation of upland cotton (Gossypium hirsutum):

[0033] Using the genetic transformation method mediated by Agrobacterium, the T66-35S-OK-Bt-PS-Tnos-2300 vector was used to transform Jimian 14 (National Cotton Medium-term Bank, acquired by China Cotton Research Institute, unified number: ZM-30270) hypocotyl.

[0034] Agrobacterium LBA4404 containing the T66-35S-OK-Bt-PS-Tnos-2300 vector was picked and inoculated to contain 50 mg / L of kanamycin (km), 50 mg / L of rifampicin (rif) and Streptomycin (streptomycin, S / Sm) 50mg / L LB liquid medium, 28 ℃ shaking dark culture overnight to the logarithmic phase of bacterial growth. Dilute the bacterial liquid with LB or YEB liquid medium at the ratio of bacterial liquid: medium 1:50 to 1:100, then shake and culture for 4 to 6 hours, and dilute the bacterial liquid to an OD600 value of 0.8 to 1.0.

[0035] The transgenic recipient material is Jimian 14. The hypocotyls of sterile seedlings grown for 3 to 4 ...

Embodiment 3

[0036] Screening and identification of embodiment 3.T0 generation transgenic material:

[0037] The Elisa detection of insect-resistant protein and the insect-resistant test were carried out at the seedling stage, flowering stage, bud stage and boll stage respectively. According to the Elisa test at the seedling stage, there were 125 transgenic cotton plants with high expression of insect-resistant protein. After the insect resistance test, there are 94 strains resistant to cotton bollworm, of which 25 strains have strong resistance to cotton bollworm.

[0038]

[0039]

[0040]

[0041]

[0042]

[0043] P+: PCR positive plants.

[0044] Insect resistance analysis: N: no resistance; R: resistance; HR / HR-: strong resistance

[0045] Combined with the results of Elisa and insect resistance test, 22 materials with strong insect resistance were selected for Southern analysis. For Southern analysis, combined with plant fertility, DNA was extracted from cotton tis...

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Abstract

The invention provides a cotton bollworm transformation event and a characteristic sequence and method for detecting primer and method. The carrying a 2 -6 transformation event cotton plants in the first group chromosome comprising said event foreign inserting DNA sequence and the cotton genome DNA sequence of engagement. By utilizing the foreign inserting DNA sequence and the flank engagement region of the cotton genome DNA sequence, a detection primer designed for aiming at a 2 -6 event specificity detection. Aiming at a 2 -6 event detection method for plant breeding by using the affair application provides convenience for tracking the specific gene is inserted into the event., and the specific gene is inserted into the event can be used as molecular markers to improve the breeding efficiency.

Description

technical field [0001] The invention belongs to the field of plant molecular biology, especially the field of transgenic crop breeding in agricultural biotechnology research, and particularly relates to cotton transformation event A2-6 with resistance to cotton bollworm and a unique method for detecting the transformation event. Background technique [0002] Worldwide, insect pests have brought great losses to agricultural production. Traditional control techniques using chemical pesticides have played a huge role in traditional agricultural practices. However, this pest control technology has great disadvantages. On the one hand, the use of a large amount of toxic chemical pesticides not only pollutes the environment, but also easily remains, which seriously threatens people's health; Resistance to pesticides, leading to outbreaks of pest infestations. For example, in the early 1990s, due to the development of drug resistance of cotton bollworms, the outbreak of cotton bo...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12N15/84C12N1/21C12Q1/68A01H1/02A01H1/04A01H5/00
CPCC12N15/8286C12Q1/68C12N5/14A01H5/10C12Q1/6895Y02A40/146
Inventor 崔洪志何云蔚王君丹陈文华王建胜宋辉平
Owner BIOCENTURY TRANSGENE CHINA
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