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Double T-DNA vector capable of achieving agrobacterium co-transformation and establishment method and application thereof

A technology of co-transformation and Agrobacterium, applied in the field of genetic engineering, can solve the problems of inability to obtain transgenic positive plants, wrong retention, and difficulty in achieving separation, and achieve the effect of simple and rapid elimination.

Inactive Publication Date: 2016-04-20
SICHUAN AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the process of in-depth research on the co-transformation method, researchers gradually realized that this method has drawbacks. First, different receptor tissues have different integration efficiencies for the selection marker gene and the target gene. T 0 Individuals containing only the target gene in the generation will be eliminated, while individuals containing only the selectable marker gene will be retained by mistake; moreover, the selectable marker gene and the target gene are easily integrated into the same position of the recipient plant genome during transformation point, and finally it is difficult to achieve the purpose of separation in the offspring
In subsequent studies, the super binary vector containing two T-DNA regions constructed by inserting the selectable marker gene and the target gene into independent T-DNA regions in the same plasmid greatly improved the selectable marker gene and target gene. co-integration efficiency, but most of the selectable marker genes and target genes are integrated into the same site in the genome so that in T 1 In the process of reproductive growth, the genes on the double T-DNA carrier cannot be separated, and it is impossible to obtain transgenic positive plants containing only the target gene

Method used

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  • Double T-DNA vector capable of achieving agrobacterium co-transformation and establishment method and application thereof
  • Double T-DNA vector capable of achieving agrobacterium co-transformation and establishment method and application thereof
  • Double T-DNA vector capable of achieving agrobacterium co-transformation and establishment method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1 Construction of Vector A Containing Two Independent Left Border LB Sequences

[0045] 1. Design primers to amplify the terminator NOS and the left border LB

[0046] According to the sequence of the published pCMBIA1302 vector (GenBank: AF234298.1), the primers for the amplification terminator NOS were designed, including restriction enzyme sites BsteII and BamHI, and the sequences were shown in SEQ ID NO: 1, SEQ ID NO: 2; The primers of the left border LB are added, and the primers include the restriction enzyme cutting sites BglII and SphI, wherein BamHI and BglII are homologous enzymes. See SEQ ID NO:3, SEQ ID NO:4 for the sequence. Primers were synthesized by Shanghai Yingjun Company.

[0047] Using pCAMBIA1302 as a template, using SEQIDNO:1 and SEQIDNO:2 as primers to carry out PCR amplification of NOS, i.e. SEQIDNO:5, the target fragment is about 273bp ( figure 1 ). Use SEQIDNO:3 and SEQIDNO:4 as primers to carry out PCR amplification LB, i.e. SEQIDN...

Embodiment 2

[0050] Embodiment 2 contains the construction of two vectors B facing away from the UBI promoter sequence

[0051] 1. Design primers to amplify two independent UBI promoter fragments

[0052] According to the sequence of the published BinaryvectorpGA1611 carrier (GenBank: AY373338.1), design the primers for amplifying two independent UBI promoter fragments, the primers are designed according to the requirements of homologous recombination primers, the sequences are shown in SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10; a restriction site SalI was introduced into primers SEQ ID NO: 8 and 9.

[0053] Using the vector containing the UBI promoter as a template, using SEQIDNO: 7 and SEQIDNO: 8 as primers to perform PCR amplification of UBI-1, i.e. SEQIDNO: 11, the target fragment is about 2050bp ( image 3 ). Use SEQIDNO:9 and SEQIDNO:10 as primers to carry out PCR amplification UBI-2, i.e. SEQIDNO:12, the target fragment is about 2063bp ( image 3 ). The amplified p...

Embodiment 3

[0056] Example 3 Construction of Vector C Containing Two Right Border RB Sequences

[0057] 1. PCR amplification of two independent RB fragments

[0058] According to the sequence (GenBank: AF234298.1) of published pCMBIA1302 carrier, design homologous recombination primer amplification two independent RB-1, RB-2 fragment, primer sequence is respectively SEQIDNO:13, SEQIDNO:14 and SEQIDNO: 15. SEQ ID NO: 16.

[0059] Using pCAMBIA1302 as a template, using SEQIDNO:13 and SEQIDNO:14 as primers to carry out PCR amplification to obtain the RB-1 fragment, the sequence number is SEQIDNO:17, and the target fragment is about 179bp ( Figure 4 ). Carry out PCR amplification with primer and SEQIDNO:15, SEQIDNO:16 to obtain the RB-2 fragment, i.e. SEQIDNO:18, the target fragment is about 173bp ( Figure 4). Gel recovery of the amplified product.

[0060] 2. Homologous recombination ligation and sequencing verification

[0061] The carrier obtained in Example 2 was carried out to Sa...

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Abstract

The invention provides a double T-DNA vector capable of achieving agrobacterium co-transformation. The double T-DNA vector is obtained by serially connecting a reporter gene GUS and a resistant selection marker gene, serially connecting a fluorescence reporter gene and a target gene, then respectively connecting the two serially connected sequences to two independent T-DNA areas and establishing the sequences to a plant binary expression vector. The vector is utilized to cultivate transgenic plants without selection markers and can quickly test positive transgenic plants from offspring.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and in particular relates to a double T-DNA carrier capable of realizing the co-transformation of Agrobacterium and its construction method and application. Background technique [0002] In today's China, the contradiction between food supply and demand is becoming increasingly prominent. As the most direct and effective technical means to improve crop varieties and improve crop yield and quality, genetic engineering technology has more and more important practical significance. [0003] Transgenic technology emerged in the 1980s. At present, this technology has become the most direct and effective technical method for high-yield, high-quality, high-resistance, and wide-ranging crop breeding. This technology has been successfully applied to the improvement of economic crops, food crops, vegetables, flowers, medicinal plants, fruit trees and pastures. The data report pointed out that in 2014, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/84C12N15/66C12N15/65A01H5/00
CPCC12N15/8205C12N15/8209
Inventor 江千涛张晓伟杨强王际睿陈国跃祁鹏飞刘亚西蒲至恩李伟魏育明郑有良
Owner SICHUAN AGRI UNIV
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