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Expression vector for deleting exogenous genes in polished rice of transgenic rice and application thereof

A technology for transgenic rice and expression vectors, applied in the application and use of vectors to introduce foreign genetic material, angiosperms/flowering plants, etc., can solve the problems of removing exogenous genes in transgenic rice polished rice, and achieve the effect of solving the problem of genetic stability

Active Publication Date: 2013-04-17
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] So far, there is no method that can effectively remove exogenous genes in transgenic rice polished rice without damage to rice

Method used

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  • Expression vector for deleting exogenous genes in polished rice of transgenic rice and application thereof
  • Expression vector for deleting exogenous genes in polished rice of transgenic rice and application thereof
  • Expression vector for deleting exogenous genes in polished rice of transgenic rice and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1: Construction of rice T-DNA transformation vector p1300-326-Ubi-YFP-EnP2-FLP

[0029] 1) Artificial synthesis and construction of recombinant system

[0030] According to Luo et al. (Lou K., et al.'GM-gene-deletor': fused loxP-FRT recognition sequences dramatically improve the efficiency of FLP or CRE recombinase on transgene excision from pollen and seed of tobacco plants.Plant Biotech.J. , 2007, 5: 263-274) the loxP-FRT sequence reported in the literature, as well as the NOS sequence, was artificially synthesized by the recombinant system loxP-FRT-FLP-Nos-loxP-FRT (base Sequence such as SEQ ID NO: 6), contains HindIII and EcoRI restriction sites at both ends of the sequence, and contains a section of multi-cloning site sequence before the FLP sequence (the restriction site of the multi-cloning sequence is ClaI-NdeI-PstI- SwaI-SalI-NcoI-BamHI-NruI-KpnI-HpaI-Bsp1407I-AscI-SnaBI-BglII-ApaI-StuI-SpeI-SacII-XhoI-SmaI), inserted into pCAMBIA1300 after digestion w...

Embodiment 2

[0057] Embodiment 2: Obtaining of transgenic rice without exogenous gene in polished rice

[0058] The method for obtaining transgenic rice is to adopt existing technology (Pan G., et al. Map-based cloning of a novel rice cytochrome P450 genes Cyp81A6 that confers resistance to two different classes of herbs. Plant Molecular Biology, 2006, 61: 933 -943), select plump rice variety Nipponbare seeds, remove the hulls, and induce callus as transformation materials. The T-DNA vector p1300-326-Ubi-YFP-EnP2-FLP constructed in Example 1 was introduced into Agrobacterium EHA105 by electric shock method. Take the Agrobacterium plate containing the T-DNA vector p1300-326-Ubi-YFP-EnP2-FLP, pick a single colony and culture it in LB medium to prepare Agrobacterium for rice transformation.

[0059] Blot the rice callus to be transformed slightly dry on sterile filter paper, put the callus in OD 600 0.5 Agrobacterium bacteria solution (containing acetosyringone, 200 μmol / L), put it at room ...

Embodiment 3

[0069] Example 3: Construction of double T-DNA vectors for breeding non-marker genes and non-Bt insect-resistant genes in polished rice

[0070] 1) Construction of the recombination system

[0071] Insert p1300-326 in Example 1 into the super double T-DNA vector pSB130 (Liu Qiaoquan, genetic engineering technology improves rice lysine content. Doctoral dissertation, 2002, Yangzhou University Library), pSB130 after HindIII and EcoRI digestion The plasmid was provided by Academician Xin Shiwen, Department of Biology, The Chinese University of Hong Kong, forming pSB326.

[0072] 2) Cloning of the endosperm-specific promoter RAG1

[0073] According to Wu et al. (Wu CY., et al. Promoters of rice seed storage protein genes direct endorsperm-specific gene expression in transgenic rice. Plant Cell Physiol., 1998, 39: 885-889 and GenBank Accession NO: D11433.1) in According to reports in the literature, two primers were synthesized (Table 5) to amplify the endosperm-specific promoter...

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Abstract

The invention discloses an expression vector for deleting exogenous genes in polished rice of transgenic rice, which comprises an original vector and a recombinant sequence inserted into the original vector, wherein the recombinant sequence comprises Lo*P-FRT elements at the two ends, an intermediate exogenous target gene expression box and a Cre gene or FLP gene expression box, and a promoter ofthe Cre gene or FLP gene expression box is an endosperm specific promoter. The invention further discloses a method for utilizing the expression vector to delete the exogenous genes in the polished rice of the transgenic rice, the obtained expression vector is transferred into rice cells, and the cultivation is performed for generating a transgenic rice plant. The transgenic rice which is obtained through the method can achieve the purposes of resisting pests and (or) resisting weedicides and the like; furthermore, endosperm, namely the polished rice does not contain the exogenous target genes, and the safety problem in eating is further solved.

Description

technical field [0001] The invention relates to the field of rice genetic engineering, in particular to an expression vector for deleting exogenous genes in transgenic polished rice and its application. Background technique [0002] Since transgenic rice was obtained for the first time in 1988 using protoplasts as recipients, rice transgenic technology has developed rapidly (Toriyama, K., et al. Transgenic rice plants after direct gene transformation into protoplasts. Bio / Technology, 1988, 6: 1072 -1074). At present, using different transgenic methods, scientists have introduced exogenous target genes such as herbicide resistance genes, insect resistance genes, disease resistance genes, stress resistance genes and different quality improvement genes into different rice varieties (lines), and obtained a large number of transgenic rice. . So far, the U.S. has approved Aventis’ bar gene-transformed herbicide-tolerant rice LL RICE 06, LL RICE 62, and LL RICE 601; the Iranian g...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/63C12N15/82A01H5/00
Inventor 潘刚张春娇王宁程方民
Owner ZHEJIANG UNIV