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A carrier suitable for gene superposition and its application

A technology of gene superposition and carrier, applied in the fields of application, genetic engineering, plant gene improvement, etc., can solve the problems of huge workload and low probability of transgenic plants, and achieve the effect of reducing workload and efficient expression

Active Publication Date: 2018-07-17
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

It can be seen that in the existing technology, the workload of obtaining transgenic plants with multiple genes superimposed is very large, and the probability is low

Method used

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  • A carrier suitable for gene superposition and its application
  • A carrier suitable for gene superposition and its application
  • A carrier suitable for gene superposition and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Embodiment 1 is suitable for the carrier of gene stacking

[0046] Vector construction employs conventional recombinant DNA methods. All PCR reactions used Phusion High-Fidelity DNA Polymerase (NEB Beijing, China). In this example, rice is taken as an example to illustrate the construction process of a vector suitable for rice gene stacking. Vectors suitable for other biological gene stacking can be similarly constructed according to this method. For example, the promoter sequence in the vector, the screening gene, etc. The characteristics of the organism are selected.

[0047] 1. Construction of pZH37 vector suitable for rice gene stacking

[0048] The construction process of pZH37 vector is as follows: figure 1 shown, combined with figure 1 The specific method of its construction is described as follows:

[0049] 1) Construction of recombinant vector pZH2

[0050] The backbone vector for constructing pZH37 comes from the vector pC13Bar of the pCambia series (f...

Embodiment 2

[0086] Example 2 The method for producing rice gene stacking lines

[0087] 1. Rice Transformation

[0088] The vectors pZH37 and pZH36 constructed in Example 1 or 2 were transformed into rice by Agrobacterium, and the rice variety was Zhonghua 11. The rice transformation method was referred to Li, M.R., Li, H.Q. (2003) A simple and highly efficient Agrobacterium mediated rice transformation system . Acta Biol Exp Sin. 36, 289-294.

[0089] 2. Screening

[0090] (1) PCR screening

[0091] A total of 3953 regenerated plants were obtained from the above Agrobacterium transformation for screening of target strains. detected by PCR gus DNA has 3136 positive transgenic plants.

[0092] (2) qPCR screening

[0093] 1) Gus qPCR Screening of Genes

[0094] For the 3136 plants screened above, real-time fluorescent quantitative PCR was used for further screening, and the specific operation was as follows: a 10ul reaction system was used, including 2 × SYBR Premix Ex Taq Ⅱ (Ba...

Embodiment 3

[0134] Example 3 Genomic loci suitable for rice gene stacking

[0135] Using the genome sequence information obtained by TAIL-PCR of the 7 strains of the present invention in Example 2, according to the genome sequence information inserted in the left and right borders of the transgenic T-DNA, use online bioinformatics software to search the rice genome database, and the target strain Mapping of T-DNA to rice chromosomes (http: / / blast.ncbi.nlm.nih.gov / Blast.cgi, http: / / rapdb.dna.affrc.go.jp / , http: / / rice.plantbiology .msu.edu / analyses_search_blast.shtml). For specific information, please refer to Sakai, H., Lee, S.S., et al., (2013) Rice Annotation Project Database (RAP-DB): An integrative and interactive database for rice genomics Plant & Cell Physiol. 54, e6; Kawahara, Y ., de la Bastide, M., Hamilton J. P., Kanamori, H., McCombie, W.R., Ouyang, S., Schwartz, D. C., Tanaka, T., Wu, J., Zhou, S., Childs, K. L., Davidson , R. M., Lin, H., Quesada-Ocampo, L., Vaillancourt, B....

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Abstract

The invention discloses a vector applicable to gene stacking and application of the vector. The vector contains an attP site or an attB site, a lox site, a screening and reporting gene and lox sites, wherein the lox site is located on one side of the attP site or the attB site, and the lox sites are located on two sides of the screening and reporting gene; RS2 sites or MRS sites are further located on two sides of a segment connected with the attP site, or the attB site, the lox site and a marking and reporting gene. By integrating the vector into a rice genome through a T-DNA sequence, a strain applicable to rice gene stacking can be generated. The rice genome applicable to the gene stacking has seven sites, and multiple target genes can be stacked in the seven sites to be efficiently expressed, so that the number of separation sites can be reduced, the workload for leading transgenosis from a laboratory strain to farmland varity is greatly reduced, the normal expression of other genes is basically not influenced, and novel genes can be stacked to existing transgenosis sites by commercial product researchers.

Description

technical field [0001] The invention relates to a carrier suitable for gene stacking and its application. Background technique [0002] Traditionally, the introduction of new genes into commercial varieties is usually through classical breeding methods to produce a homozygous line, which must include not only the transgene, but also the excellent traits of field varieties. In diploid plants, in the absence of genetic linkage, the proportion of lines homozygous for n independent traits by segregation is (¼) n . Therefore, if you want to obtain homozygous lines with 7 excellent traits and 1 transgenic trait, you need more than 16,000 individual plants to obtain, the ratio is (¼) 7 . If there are 3 transgenic loci, then the proportion of homozygous lines is (¼) 10 , it is possible to screen homozygous lines among more than 1,000,000 individual plants. Due to the large number of region-specific varieties and numerous breeding objectives for local field traits, adding segre...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/82C12N5/10C12N15/84C12N15/11A01H5/00A01H6/46
Inventor 区永祥韩志国
Owner SOUTH CHINA BOTANICAL GARDEN CHINESE ACADEMY OF SCI