Double T-DNA carrier and its application in cultivating of non selecting sign transgene rice

A technology of transgenic rice without selection marker, applied in recombinant DNA technology, application, genetic engineering and other directions, can solve the problems of double T-DNA vector, such as large molecular weight, limited effect, and lack of versatility.

Inactive Publication Date: 2005-03-23
YANGZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Because: (1) Taking marker gene knockout systems based on recombinases, transposons or intrachromosomal recombination as examples, although these systems have been proven in model plants such as tobacco and Arabidopsis, so far there are very few See reports on important food crops such as rice and corn; secondly, the probability of homologous recombination or transposition in rice and other crops is often not high, and these systems still need to be combined with genes containing recombinase or transposase Transgenic plant hybridization and genetic segregation of subsequent generations, so the effect of marker-free transgenic plants tha

Method used

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  • Double T-DNA carrier and its application in cultivating of non selecting sign transgene rice
  • Double T-DNA carrier and its application in cultivating of non selecting sign transgene rice
  • Double T-DNA carrier and its application in cultivating of non selecting sign transgene rice

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1: Construction of Plant Double T-DNA Universal Binary Vectors pSB130 and pSB131

[0033] 1.1 According to the nucleotide sequence of the binary vector pCAMBIA1300 (CAMBIA, Australia), two primers LBP were designed 1 (5'-CAA GCGGCCGC GAGATCATCCGTGTTT-3') and LBP 2 (5'-GTA GAATTC GACCGGATCTGTCGATCGA-3') to amplify the left border sequence of its T-DNA region from the binary vector. Not I and Eco RI (underlined) enzyme cutting sites were added to the 5' ends of these two primers, wherein LBP 2 It is located on the side of the left border sequence close to the T-DNA. The conditions of the PCR reaction were 95°C, 5min; 95°C, 50sec, 55°C, 50sec, 72°C, 30sec, 30 cycles; 72°C, 7min. The 495bp PCR product was digested with Not I and Eco RI and cloned into the intermediate vector pBluescript SK - In , the identified positive clone is called pBSK / LB.

[0034] 1.2 Digest the binary vector pCAMBIA1300 with Hind III and Eco RI, fill it up with a large Klenow fragmen...

Embodiment 2

[0039] Example 2: Utilizing double T-DNA vectors to cultivate high-quality transgenic rice with high lysine content without selection markers

[0040] 2.1 Construction of double T-DNA binary vector carrying high lysine content protein LRP gene

[0041] The lack of lysine in rice is considered to be the first limiting essential amino acid in rice. Using transgenic technology to express heterologous lysine-rich proteins in rice can provide the total lysine content in rice. The high lysine content protein (Lysine-rich protein, LRP) gene cloned from the square bean contains 10.7% lysine (Sun et al., USA patent, 1998) in its coded protein, is an ideal for Genes for improving the nutritional quality of rice. In order to express it specifically and highly in the endosperm of transgenic rice seeds, the cDNA encoding LRP was connected to the glutelin gene Gt1 promoter of rice itself, and then cloned into the double T-DNA binary vector pSB130( figure 1 -A) In the multiple cloning site...

Embodiment 3

[0062] Example 3: Utilizing double T-DNA vectors to cultivate insect-resistant transgenic Bt rice without resistance selectable markers

[0063] 3.1 Construction of the double T-DNA binary vector carrying the Bt toxin gene

[0064] The Bt toxin gene derived from Bacillus thuringiensis is currently the most widely used insect resistance gene, and the Bt toxin gene has been successfully used in cotton, corn, etc.; the gene introduced into rice has good resistance to rice borer. In this example, the Bt poisonous protein gene is resynthesized according to plant preferred codons, which can make the gene more efficiently and stably expressed in transgenic plants; in addition, a transit peptide signal sequence from plants is added to the N-terminal of the Bt poisonous protein , the expression product can be localized in the endoplasmic reticulum, making the expression of the target protein more efficient (Peng Rihe et al., Acta Biochemistry and Biophysics, 2001, 33: 219-224). The te...

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Abstract

The invention provides a simple and convenient Agrobacterium Tumefacies dual carrier containing double T-DNA structural regions, and a method of using the carrier system to cultivate transgenic rice without resistance selection label. With the help of the principle of co-conversion mediated by root nodule Agrobacterium Tumefacies, the system contains two separate T-DNA structural sections, where the first T-DNA region contains antibiotic resistance selection label gene and the second one contains a universal polyclonal site able to be arbitrarily inserted with destination gene. The double-T-DNA carrier has small molecular weight, easy to clone and after the destination gene and necessary regulation and control series are cloned in the T-DNA region containing the polyclonal site, it realizes rice co-conversion; by selfing, it selects transgenic individual with destination gene but without selectin label gene from the self-bred progeny, thus eliminating the negative effect on transgenic plant commercialized production, etc, possibly caused by selection label gene.

Description

technical field [0001] The invention relates to a method for cultivating transgenic crops without a selection marker for resistance, and belongs to the application technology in the field of biology and modern agricultural technology. More specifically, the present invention relates to a binary vector with two independent T-DNA structural domains, respectively containing a resistance selectable marker gene and a target gene; relates to the use of the double T-DNA binary vector system to generate simultaneously containing selection After the co-transformation of transgenic plants with the marker gene and the target gene, individuals containing only the target gene but no resistance selectable marker gene are selected and bred in the offspring population, thereby eliminating the possible impact on the commercial production of transgenic crops due to the existence of the selectable marker. coming negative impact. Background technique [0002] Since the first transgenic plant e...

Claims

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Application Information

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IPC IPC(8): C12N15/65C12N15/82C12N15/84
Inventor 刘巧泉辛世文于恒秀顾铭洪
Owner YANGZHOU UNIV
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