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Wild eggplant cytochrome oxidase gene StCYP77A2 as well as expression vector and application thereof

An expression vector, 1.stcyp77a2 technology, applied in the field of plant genetic engineering, can solve problems affecting crop yield and quality, economic loss, etc.

Inactive Publication Date: 2013-07-24
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The pathogen is widely distributed and has many hosts, such as upland cotton, eggplant, tomato, potato, cabbage, rape, cabbage, pepper, etc., which seriously affects the yield and quality of these crops, causing huge economic losses

Method used

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  • Wild eggplant cytochrome oxidase gene StCYP77A2 as well as expression vector and application thereof
  • Wild eggplant cytochrome oxidase gene StCYP77A2 as well as expression vector and application thereof
  • Wild eggplant cytochrome oxidase gene StCYP77A2 as well as expression vector and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1 Cloning of Wild Eggplant Torubum StCYP77A2 Gene

[0028] According to the analysis of the mRNA sequence of the CYP77A2 gene of eggplant cultivars in GenBank, the primers CYP77-s including the complete open reading frame were designed: (5'-ATGGATTTTTTCTCAACTTCCT-3', SEQ ID NO.3); CYP77-a: (5'-ATTCTTGGTTTAATTTTAGCTCT-3', SEQ ID NO. 4)). Use the first strand of cDNA of wild eggplant young leaves as a template to perform RT-PCR. The reaction system is 25ul. The PCR cycle program is as follows: 94°C for 5 minutes; 94°C for 50 seconds, 62°C for 40 seconds, and 72°C for 50 seconds (35 cycles in total cycle); 72°C for 10 minutes. A fragment of about 1600bp was obtained by sequencing. After electrophoresis on 1.0% agarose, the PCR product was recovered with the gel recovery kit of Bao Bio Company, connected to pMD18-T Vector, transformed into Escherichia coli DH5α, single clone was detected by PCR, and sequenced. The sequencing results were analyzed with DNAssist so...

Embodiment 2

[0029] The construction of embodiment 2 wild eggplant Torubum StPP5 gene overexpression vector ( figure 2 )

[0030] Remove the TAA stop codon from the full-length cDNA of the cloned gene StCYP77A2, and connect it to the overexpression vector PCB2008E (Zhi-Yong Lei, Ping Zhao et al. (2007) High-throughput Binary Vectors for Plant Gene Function Analysis, Journal of Integrative Plant Biology49(4):556-567) at the multiple cloning site behind the 35S promoter, and was fused with the reporter gene GFP protein. The recombinant vector was transformed into Escherichia coli DH5α, positive monoclonal colonies were picked by PCR, shaken and plasmids were extracted, and sequenced. Sequencing results showed that the target gene had been successfully inserted into the PBC2008E vector, and the recombinant plasmid was named PCB2008E-StCYP77A2. Specific steps are as follows:

[0031] Add Hind III and BamH I restriction sites to the cDNA forward and reverse ends of the StCYP77A2 gene (witho...

Embodiment 3

[0035] Example 3 Obtaining Transgenic StCYP77A2 Gene Tobacco Using Agrobacterium-mediated Transformation

[0036] Using the gene transformation method mediated by Agrobacterium, tobacco cotyledons were pre-cultured for 2 days, then infected with Agrobacterium suspension for 5 minutes, and co-cultured on the co-cultivation medium for 2 days. After the tobacco leaf explants were infected with Agrobacterium, they were transferred to the callus differentiation medium, and callus tissue was produced in about 15 days, and adventitious buds appeared in about 20 days, and then all the formed adventitious buds were transferred to the culture medium containing 20 mg / L Hyg B Secondary screening was carried out in the MS medium, and the non-transformed shoots eventually died of albinism because they had no antibiotic resistance. Subsequently, the obtained positive plants were transferred to MS medium for propagation, verified by PCR and southern, and finally transgenic tobacco plants were o...

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Abstract

The invention relates to wild eggplant cytochrome oxidase gene StCYP77A2 as well as an expression vector and an application thereof. CYP77A2 gene is cloned from eggplant for the first time, and named as StCYP77A2; a plant expression vector is constructed, and transferred in cultivated-species tobacco by virtue of an agrobacterium-mediated method to obtain transgenic tobacco plants; and the resistance to greensickness of the verified transgenic plants is further analysed via a root-irrigation method, and the result indicates that, after greensickness bacteria are inoculated, the morbidity and the disease index of overexpression for the transgenic tobacco of the StCYP77A2 are 88.89% and 52.78, and lower than 100% and 77.78 of a reference respectively, so that the gene has an inhibition effect on greensickness to a certain extent. Therefore, the gene StCYP77A2 can be applied to anti-greensickness breeding for crops such as eggplant as a resistance gene resource.

Description

technical field [0001] The invention relates to wild eggplant cytochrome oxidase gene StCYP77A2 and its expression carrier and application, belonging to the field of plant genetic engineering. Background technique [0002] Cytochrome P450 (Cytochrome P450, hereinafter referred to as CYP450) is a kind of heme oxidase system, which is ubiquitous from simple bacteria to higher animals and plants. many important metabolic processes. Studies have shown that its three-dimensional structure is very conservative, and the classic CYP450 has a highly conserved FXXGXRXCXC domain in the catalytic center. P450 has great potential for application in plant disease resistance engineering, such as: 1) Inhibition of gene activity: P450 (CYP71D16) with hydroxylase activity isolated from tobacco hairy glands, when using antisense and co-suppression technology to identify its function, found that The substrate (cembratrieneol) catalyzed by CYP71D16 increased significantly, which made the plant...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/53C12N9/02C12N15/82A01H5/00
Inventor 杨清史策陈敏决登伟
Owner NANJING AGRICULTURAL UNIVERSITY
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