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Indica rice target line for gene stacking of specific sites mediated by recombinase

A technology of recombination sites and recombinases, applied in the field of indica rice target lines, can solve problems such as genetic transformation difficulties, achieve the effects of reducing separation sites, reducing breeding workload, and reducing release evaluation costs

Active Publication Date: 2020-05-08
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 generally difficult, and the transformation efficiency is usually around 5%.

Method used

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  • Indica rice target line for gene stacking of specific sites mediated by recombinase
  • Indica rice target line for gene stacking of specific sites mediated by recombinase
  • Indica rice target line for gene stacking of specific sites mediated by recombinase

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Embodiment 1 is suitable for the carrier of gene stacking

[0045] The structures of vectors pZH109, ​​pZH108 and pZH93 are as follows figure 1 As shown, its construction refers to the standard DNA recombination method. All PCR reactions used Phusion high-fidelity polymerase (NEB Beijing, USA). The sugarcane baculovirus (ScBV) promoter and terminator octopine synthase (ocs) control the initiation and termination of the green fluorescent reporter gene gfp, respectively. The rice actin promoter and terminator cauliflower virus (CaMV35s) control the initiation and termination of the hygromycin gene hpt, respectively. The CaMV 35S promoter and terminator control the initiation and termination of the herbicide resistance gene bar, respectively. The promoter of the cre recombinase gene is the CaMV35S promoter, and the terminator is the nopaline synthase (nos) terminator.

[0046] This application adopts two strategies to obtain the starting target line of indica superposi...

Embodiment 2

[0049] The screening of embodiment 2 initial target lines

[0050] method:

[0051] Agrobacterium-mediated transformation

[0052] About 2000 immature embryos of indica rice Huanghuazhan or 500 indica rice R900 were infected with Agrobacterium. One part was infected with Agrobacterium EHA105 containing the target line vector pZH109, ​​and the other part was co-transformed with Agrobacterium containing pZH108 and Agrobacterium containing pZH93. For specific transformation methods, please refer to Hiei Y. and Komari T.2008. Agrobacterium-mediated transformation of rice using immature embryos or calli induced from mature seed. Nature Protocol (3) 824–834.

[0053] GFP observation

[0054] GFP expression was observed by a Leica inverted fluorescence microscope DMI6000B (Leica, Wetzlar, Germany). The excitation filter wavelength range used is 440-520nm, and the barrier filter wavelength is 510LP.

[0055] PCR analysis

[0056] About 100 mg of fresh or frozen GFP-expressed you...

Embodiment 3

[0091] Example 3 Initial Target Line Genome Location Map

[0092] figure 1 The insertion position shown in C is obtained by comparing the left and right side DNA sequences with the rice genome database (Wang, J., Kong, L., Zhao, S., Zhang, H., Tang, L., Li, Z., Gu , X., Luo, J., and Gao, G.2011. Rice-Map: a new-generation rice genome browser. BMC Genomics.12, 165) comparison and determination. For detailed DNA sequence see Figure 4 and Figure 5 . Important features of each insertion site are listed here (insertion sites are figure 1 A and 1B3 are described from the T-DNA left border to the right border), that is, five genome sites suitable for indica rice gene stacking of the present invention:

[0093] The target site of the target line H11: located between 24,920,581 and 24,921,595 of the long arm of the 12th chromosome, the 21bp genome sequence from 24,921,594 to 24,920,581 was deleted. At the left end of the T-DNA, the first 24 bp of the 26 bp LB was deleted. At t...

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Abstract

The present invention discloses five starting target lines of gene stacking of specific sites mediated by recombinase suitable for indica rice. Each starting target line has an accurate single copy transgenic DNA, the DNA does not affect endogenous genes, an insertion site is at least 1 kb away from a nearest gene coding region, the DNA is far away from centromere, and a reporter gene gfp is wellexpressed. The target line contains attP and Lox recombination sites, which is conducive to stacking relevant functional genes on the gene sites through site-specific recombination fixed-point stacking. Therefore, the number of isolation sites can be reduced, and breeding workload of transgenic infiltration from experimental strains into local fine varieties can be greatly reduced. In addition, commercial product developers can also integrate the existing transgenic sites, thereby greatly reducing cost of release evaluation.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to an indica rice target line for gene stacking of specific sites mediated by recombinases. Background technique [0002] Transgenic technology is the use of traditional breeding methods to introduce commercial varieties. 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 / ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82A01H5/00A01H6/46
CPCC12N15/8213
Inventor 区永祥李如玉
Owner SOUTH CHINA BOTANICAL GARDEN CHINESE ACADEMY OF SCI
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