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Expression vector of duplicate inverted waxy gene, preparation method, and application

A waxy gene and expression vector technology, applied in the fields of genetic engineering and plant genetic engineering, can solve the problems of insignificant reduction of related gene expression, unfavorable safety control of genetically engineered plants, and difficulty in obtaining transformants.

Inactive Publication Date: 2007-05-09
SHANGHAI NORMAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

[0010] The technical problem to be solved by the present invention is to provide a double antisense waxy gene expression vector and its preparation method and application, to overcome the existing expression vector containing resistance selection marker gene or containing a promoter from a virus, It is not conducive to the safety control of genetically engineered plants; and only contains one antisense waxy gene, the related gene expression of the obtained transformants and their offspring is not significantly reduced, and it is difficult to obtain homozygous for both T-DNA integration sites Defects in transformants

Method used

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  • Expression vector of duplicate inverted waxy gene, preparation method, and application
  • Expression vector of duplicate inverted waxy gene, preparation method, and application
  • Expression vector of duplicate inverted waxy gene, preparation method, and application

Examples

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Embodiment 1

[0052] This example is to illustrate the method for constructing a double antisense gene expression vector and the structural composition of the obtained vector.

[0053] Methods such as conventional PCR amplification, restriction endonuclease digestion and ligation involved in the operation of constructing the expression vector mainly refer to "Molecular Cloning Experiment Guide" (Sambrook J et al., Molecular Cloning Experiment Guide (Second Edition), Science Press, 1992). For the experimental methods that do not specify specific conditions in the examples, usually follow the conventional conditions or the conditions suggested by the manufacturer.

[0054] From the published Nipponbare rice genome, search for a DNA sequence with a length of about 100 bp at the two ends of the BamHI restriction site and no SmaI and XhoI restriction sites in the nucleotide sequence, and design primers based on the sequence, with primers at both ends Carries SmaI restriction site and XhoI restr...

Embodiment 2

[0062] This example is to illustrate how to transform rice using the constructed expression vector of the double antisense waxy gene.

[0063] The Agrobacterium tumefaciens cultivation, rice transformation and resistant callus selection and plant regeneration involved in the cultivation of transgenic rice are mainly carried out according to the method of Liu Qiaoquan (Liu Qiaoquan et al., Journal of Plant Physiology, 1998, 24(3): 259-271) .

[0064] Using the co-transformation method mediated by Agrobacterium tumefaciens (Li Jianyue et al., Science Bulletin, 2004, 49(24): 2556-2561) to introduce the p13AW-2 expression vector into rice such as Shangshida No. 2 or Chao 2-10 The rice line or variety with an amylose content of about 15% can obtain a new type of germplasm with low amylose content that has no resistance selection marker gene, can be planted in the middle and lower reaches of the Yangtze River.

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Abstract

This invention discloses a method for preparing an expression vector containing double antisense waxy genes and its application. The structure and characteristics of the expression vector are shown in figure (d). The expression vector comprises promoter sequence, antisense DNA fragment of the target gene, terminator sequence, and endonuclease-sensitive sequence or marker gene sequence. All the sequences are doubled along the same direction. The expression vector can be used for breeding Gramineae plants such as rice without resistance-screening gene and with low amylase content.

Description

technical field [0001] The invention belongs to the field of genetic engineering, more specifically relates to the field of plant genetic engineering. Background technique [0002] In recent years, with the improvement of people's living standards and the development of foreign trade, the eating quality of rice has increasingly attracted people's attention. Now the research on improving the eating quality of rice has become a hot spot in the research of rice quality. According to literature reports, rice amylose content is one of the most important indicators in affecting the eating quality of rice (Li Xiulan et al., China Agricultural Science Bulletin, 2002, 18 (3): 61-64, 119; , 2002, 16(2): 157-161; Luo Zhixiang et al., China Agricultural Science Bulletin, 2002, 18(6): 18-21). Rice with higher amylose content has poorer taste; rice with lower amylose content has less swelling and better taste. [0003] A kind of novel rice that is rising in the world now---the research ...

Claims

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Application Information

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IPC IPC(8): C12N15/63C12N15/52C12N15/82
Inventor 李建粤杨丽君张伟
Owner SHANGHAI NORMAL UNIVERSITY
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