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Establishment of cotton gene superposition target system and application thereof

A cotton genome and target line technology, applied in application, genetic engineering, angiosperm/flowering plants, etc., can solve the problems of low efficiency and long cycle, and achieve the effect of reducing the number and workload

Active Publication Date: 2020-05-26
SOUTH CHINA BOTANICAL GARDEN CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, its genetic transformation is a bottleneck, the cycle is long (transformed seedlings are obtained in an average of 1 year), and the efficiency is low (average ~ 5%)

Method used

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  • Establishment of cotton gene superposition target system and application thereof
  • Establishment of cotton gene superposition target system and application thereof
  • Establishment of cotton gene superposition target system and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] The acquisition of embodiment 1 cotton initial target line (target site)

[0047] vector construction

[0048] The structure of target vector and overlay vector is as follows figure 1 and 2 As shown, its construction refers to the standard recombinant DNA method. All PCR reactions used KOD FX high-fidelity polymerase (TOYOBO, Japan).

[0049] Promoter and terminator of target vector gene:

[0050] The promoter of npt is the cotton ubiquitin (Ubiquitin) promoter, and the terminator is the cauliflower virus (CaMV35s) terminator. The promoter of the green fluorescent reporter gene gfp is the sugarcane baculovirus (ScBV) promoter, and the terminator is the octopine synthase (ocs) terminator. The promoter of the cre recombinase gene is the CaMV35S promoter, and the terminator is the nopaline synthase (nos) terminator.

[0051] Promoters and terminators for stacking vectors:

[0052] The promoter of the screening marker gene bar, bar-DsRed or DsRed-bar is CaMV 35S, and...

Embodiment 2Bxb1

[0102] Example 2Bxb1 mediates the site-specific integration of the initial target line of cotton

[0103] We tested Bxb1 recombinase-mediated site-directed integration in cotton. As indicated by the application of these target systems Figure 1 Similarly, the embryogenic callus induced by the initial target lines CTS1 and CTS4 was used as explants to test the site-specific integration of the target lines. The stacking vector contains a combination of different cotton Verticillium wilt resistance gene trait genes, and the vector size ranges from 7.6kb to 11kb ( Figure 7 A) Not equal, the stacking vector and the Bxb1 recombinase expression vector pYQ78 co-transform the embryogenic callus of the target line, and the trait gene is integrated into the attP site of the genome ( Figure 7 B), the site will be combined with the stacking vector ( Figure 7 Recombination occurs at either of the two attB sites in A), resulting in Figure 7 The structure after site-specific integrati...

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Abstract

The invention discloses an obtaining method of a cotton starting target line and an application method thereof. The cotton starting target line containing attP and Lox recombination sites obtains genome sites of four cotton starting target lines beneficial to target gene superposition through target vector transformation of cotton hypocotyls, molecular analysis and BLAST positioning analysis. Theinvention discloses the four genome sites suitable for fixed-point superposition of cotton genes. According to the present invention, with the method, the site-specific recombination site-specific superposition of the related functional gene at the gene site is easily achieved, such that the number of the separation sites can be directly reduced, the workload of the penetration of the transgene into the local field variety from the laboratory strain is substantially reduced, and the normal expression of other genes is hardly affected.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to the establishment and application of a cotton initial target line. Background technique [0002] In the prior art, transgenes have been introduced into commercial varieties through traditional breeding methods. However, hybridization between different varieties can lead to heterozygosity of many important agronomic trait genes. For breeding, a breeding line must be homozygous for traits, not only transgenic traits, but also all other good traits related to field elite varieties. For diploid and diploid-like allopolyploid plants, the proportion of lines homozygous for n independent traits by segregation in the absence of genetic linkage is (1 / 4) n . For example, the probability of obtaining homozygous lines with 6 excellent traits and 1 transgenic trait is (1 / 4) 7 , but if 3 more transgenic loci are added, then the proportion of homozygous lines obtained is (1 / 4) 10 ,...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82A01H5/00A01H6/60
CPCC12N15/8213
Inventor 区永祥李如玉李亚梅
Owner SOUTH CHINA BOTANICAL GARDEN CHINESE ACADEMY OF SCI
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