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Method for cultivating anti-cyanosis transgenic cotton and special expression vector thereof

An expression vector, a technology for verticillium wilt, which can be used in the introduction of foreign genetic material using vectors, genetic engineering, plant genetic improvement, etc., and can solve problems such as lack of in-depth understanding of plant disease resistance mechanisms.

Active Publication Date: 2010-10-27
上海中科荃银分子育种技术有限公司 +1
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AI Technical Summary

Problems solved by technology

For a long time, people have done a lot of work on the mechanism of plant disease resistance and its application in production, and accumulated a lot of information. However, due to the lack of in-depth understanding of the mechanism of plant disease resistance, disease-resistant breeding has always been It is one of the major problems in agricultural production

Method used

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  • Method for cultivating anti-cyanosis transgenic cotton and special expression vector thereof
  • Method for cultivating anti-cyanosis transgenic cotton and special expression vector thereof
  • Method for cultivating anti-cyanosis transgenic cotton and special expression vector thereof

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Experimental program
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Effect test

Embodiment 1

[0026] The construction of embodiment 1 expression vector

[0027] 1. Contains SPGAFP 1 Construction of high-efficiency expression vector

[0028] Synthetic SPGAFP 1 gene, SPGAFP 1 The nucleotide sequence of is sequence 1 in the sequence listing. SPGAFP 1 Connected to pUCm-T (purchased from Shanghai Sangon Bioengineering Co., Ltd.) to construct vector pUCm-SPGAFP 1 .

[0029] Digest pUCm-SPGAFP with XbaI and SacI respectively 1 and pBI221 (purchased from Clontech Laboratories, Inc.), the SPGAFP after digestion 1 The fragment was ligated to the pBI221 vector that cut the 1.9Kb GUS fragment with the same enzyme to obtain the intermediate vector pBI35S-SPGAFP 1 .

[0030] Digest intermediate vector pBI35S-SPGAFP with HindIII and SacI 1 get 35S-SPGAFP 1 Fragment, pCambia2300 (purchased from Cambia Laboratories Company) was digested with the same enzyme, and the two were connected to obtain the expression vector pCambia2300-35S-SPGAFP 1 . Bacteria and plant resistance ...

Embodiment 2

[0031] Example 2 will contain SPGAFP 1 The high-efficiency expression vector was introduced into cotton to obtain SPGAFP 1 cotton

[0032] Transformation of SPGAFP by Agrobacterium-mediated method 1 cotton

[0033] 1) the expression vector pCambia2300-35S-SPGAFP obtained in Example 1 1 Transform into Agrobacterium tumefaciens LBA4404 (purchased from Invitrogen Company), construct SPGAFP-containing 1 Expression vector Agrobacterium tumefaciens LBA4404 / pCambia2300-35S-SPGAFP 1 .

[0034] Pick LBA4404 / pCambia2300-35S-SPGAFP 1 A single colony of Agrobacterium was inoculated in 20ml of YEP medium containing 50mg / L kanamycin, 50mg / L rifampicin and 30mg / L streptomycin, and cultured overnight at 28°C and 280rpm until the OD600 was 0.7-0.8. Then centrifuge at 5000rpm, discard the supernatant, and resuspend the pellet with MS liquid medium. Finally, the upland cotton "Zhongmian Institute 24" (Yu Ya, Liu Chuanliang, Ma Zhiying, Li Fuguang, Li Fenglian, Wang Yufen, Wu Zhixia, Zhang ...

Embodiment 3

[0040] Embodiment 3 transfer pCambia2300-35S-SPGAFP 1 Screening of Cotton Progeny and Detection of Verticillium Wilt Resistance

[0041] 1. Preliminary screening of SPGAFP by kanamycin 1 cotton offspring

[0042] When the transformed cotton seedlings obtained in Example 2 grew 3 to 4 true leaves, the blades were smeared with kanamycin and transformed into SPGAFP 1 The preliminary screening experiment of cotton, the specific steps are as follows: apply kanamycin with a concentration of 1.0% on the surface of seedling leaves, observe after 5 days, the results show that some of the SPGAFP 1 The smear spots on the leaves of individual cotton plants turn yellow, and these individual plants with yellow leaves are symptomatic individual plants. Eliminate these symptomatic single plants, and continue the second round of screening and elimination of the remaining single plants, so that they are smeared three times in a row, and 36 T0-transferred SPGAFP strains are selected for three...

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Abstract

The invention discloses a method for cultivating anti-cyanosis transgenic cotton and a special expression vector thereof. The method for cultivating the anti-cyanosis transgenic cotton comprises the following steps: introducing the protein-coding genes of an SPGAFP1 protein into a target plant to obtain a transgenic plant with the cyanosis resistance higher than that of the target plant, wherein the amino acid sequence of the SPGAFP1 protein is sequence 2 in a sequence table; the sequence of the protein-coding genes of the SPGAFP1 protein can be sequence 1 in the sequence table; and the target plant and the transgenic plant are dicotyledon, i.e. cotton, preferably the CCRI 24. Experiments prove that the SPGAFP1 cotton obtained by introducing the expression vector into the cotton can effectively resist fungi initiating cyanosis, thus indicating that the existence of signal peptide greatly enhances the disease resistance of the transgenic plant.

Description

technical field [0001] The invention relates to a method for cultivating transgenic cotton with high Verticillium wilt resistance and a special expression carrier thereof in the biological field. Background technique [0002] Diseases have always been an important issue that cannot be ignored in agricultural production. It is estimated that the current losses caused by diseases are generally more than 10% of crop yields. Some diseases, such as rice bacterial blight, wheat scab and cotton verticillium wilt, are the main diseases of rice, wheat and cotton in my country. Afterwards, crop yields are generally reduced by 10-30%, and in severe cases, yields are reduced by 50% or even more. At the same time, it can also lead to a decline in the quality of agricultural products and cause major losses to production. At the same time, the current monoculture of agricultural production directly reduces the genetic diversity of plants, increases the continuity of time and space, and pro...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/29C12N15/63C12N1/15C12N1/19C12N1/21C12N5/10C07K14/415A01H5/00
Inventor 王义琴储成才孙勇如陈晨
Owner 上海中科荃银分子育种技术有限公司
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