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Establishment and Application of Cotton Gene Stacking Target Line

A cotton genome and cotton technology, applied in the fields of application, genetic engineering, plant genetic improvement, etc., can solve the problems of low efficiency and long cycle, and achieve the effect of reducing the number and workload

Active Publication Date: 2022-07-22
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 and Application of Cotton Gene Stacking Target Line
  • Establishment and Application of Cotton Gene Stacking Target Line
  • Establishment and Application of Cotton Gene Stacking Target Line

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1 Acquisition of cotton initial target line (target site)

[0047] Vector construction

[0048] The structure of the target vector and the superimposed vector is as follows figure 1 and 2 As shown, it was constructed with reference to standard recombinant DNA methods. All PCR reactions used KOD FX high-fidelity polymerase (TOYOBO, Japan).

[0049] Target vector gene promoter and terminator:

[0050] The promoter of npt is cotton ubiquitin (Ubiquitin) promoter, and the terminator is cauliflower virus (CaMV35s) terminator. The promoter of the green fluorescent reporter gene gfp is sugarcane baculovirus (ScBV) promoter, and the terminator is 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 selection marker gene bar, bar-DsRed or DsRed-bar is CaMV 35S, and the t...

Embodiment 2Bxb1

[0102] Example 2Bxb1 mediates site-directed integration of cotton initial target lines

[0103] We tested Bxb1 recombinase-mediated site-directed integration in cotton. As shown in the application of these target systems Figure 1 Similarly, the embryogenic callus induced by the starting target lines CTS1 and CTS4 were used as explants to test the site-directed integration of the target lines. The stacking vector contains a combination of different cotton verticillium wilt resistance genes, and the vector size ranges from 7.6kb to 11kb ( Figure 7 A) Different, the stacking vector and Bxb1 recombinase expression vector pYQ78 co-transformed embryogenic calli of the target line, and the trait gene was integrated into the genome attP site ( 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 Structure after site-directed integration of two trait genes C and 7D, structur...

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PUM

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Abstract

The invention discloses the acquisition of cotton initial target lines and an application method thereof. The initial target lines containing attP and Lox recombination sites are transformed into cotton hypocotyls through target vectors. Molecular analysis and BLAST positioning analysis are beneficial to the superposition of target genes. The genomic loci of the 4 cotton starting target lines. The four genomic loci suitable for site-specific stacking of cotton genes disclosed in the present invention are beneficial for site-specific recombination of related functional genes and site-specific stacking at the gene site, which not only directly reduces the number of separation sites, but also greatly reduces the number of isolated sites. The amount of work involved in introgressing the transgene from a laboratory line into a local field variety with little or no effect on the normal expression of other genes.

Description

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

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/82C12N15/29A01H5/00A01H6/60
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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