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A method for cultivating resistant plants and its special protein and gene

A gene, plant technology, a method applied to plants and the field of special proteins and genes, can solve problems such as photosynthesis decline, plant damage, death, and cessation

Inactive Publication Date: 2016-08-03
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, when the plant is in adversity, the cells will produce a large amount of ROS, which will increase the ROS in the whole cell
Therefore, the scavenging enzymes in chloroplasts cannot meet the requirement of removing ROS in chloroplasts, especially APX enzymes are still inactivated under adversity, resulting in damage to chloroplasts, resulting in the decline and cessation of photosynthesis, and the plants may suffer death

Method used

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  • A method for cultivating resistant plants and its special protein and gene
  • A method for cultivating resistant plants and its special protein and gene
  • A method for cultivating resistant plants and its special protein and gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0072] Embodiment 1, discovery of cotton GhSOD1 protein and its coding gene, GhCAT1 protein and its coding gene

[0073] 1. Extraction of cotton total RNA and amplification of EST target fragments

[0074] 1. Materials and reagents

[0075] The cotton material is the cotton variety "Zhongmiansuo 35", donated by Mr. Liu Zhengde of Henan Kerun Biotechnology Co., Ltd. Escherichia coli DH5α, 5'-FullRACEKit, 3'-FullRACEKit and SYBRPremixExTaq were all purchased from TaKaRa Company. pGEM-TEasyVectorSystem was purchased from Promega. RevertAidHMinusFirstStrandcDNASynthesisKit was purchased from Fermentas. GenClean Column Agarose Gel DNA Recovery Kit was purchased from Shanghai Jierui Bioengineering Co., Ltd.

[0076] 2. Extraction of cotton total RNA and amplification of EST target fragments

[0077] Soak the seeds of the cotton variety "Zhongmiansuo 35" in sterile water, and then place them in a light incubator at 28°C for 48 hours (the photoperiod is 12 hours of light / 12 hours...

Embodiment 2

[0097] Embodiment 2, construction of recombinant plasmid

[0098] 1. Construction of recombinant plasmid pBSOD

[0099] The artificially synthesized double-stranded DNA molecule shown in Sequence 1 of the Sequence Listing was inserted between the BamHI and SalI restriction sites of the pBin438 vector to obtain the recombinant plasmid pBSOD.

[0100] The double-stranded DNA molecule shown in sequence 1 of the sequence listing is named GhtSOD1 gene, and the GhtSOD1 protein shown in sequence 3 of the coding sequence listing (in sequence 3, the 1st to 82nd amino acid residues from the N-terminal form the chloroplast signal peptide, The 85th-236th amino acid residues constitute the GhSOD1 protein).

[0101] The schematic diagram of the element structure of the recombinant plasmid pBSOD is shown in figure 1 A (RB: right border; NPTII: neomycin sulfate gene; DE-35SP: double enhancer CaMV35S promoter; Nos: terminator; LB: left border; TP: chloroplast signal peptide).

[0102] 2. C...

Embodiment 3

[0106] Embodiment 3, the acquisition and identification of transgenic cotton

[0107] 1. Obtaining genetically modified cotton

[0108] 1. The recombinant plasmid pBSOD was introduced into Agrobacterium tumefaciens LBA4404 by electric shock method to obtain recombinant Agrobacterium A.

[0109] 2. Obtaining of transgenic GhtSOD1 plants

[0110] The recombinant Agrobacterium A was introduced into the cotton variety "Zhongmiansuo 35" through the Agrobacterium-mediated hypocotyl method, and the segment between RB and LB was integrated into the cotton genome through homologous recombination to obtain T 0 Substitute GhtSOD1 gene plants. T 0 T 1 On behalf of said.

[0111] The Agrobacterium-mediated hypocotyl method is to cut off the hypocotyl of the cotton variety "Zhongmiansuo 35" and co-culture it with the recombinant Agrobacterium A. For details, refer to the literature: WuHorchRB.

[0112] The PCR identification method of transgenic GhtSOD1 gene plant is as follows: take ...

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Abstract

The invention discloses a method for cultivating a resistant plant, and special protein and gene thereof. GhSOD1 protein provided by the invention is protein described as (a) or (b): (a) protein formed by 85th to 236th amino acid residues from N end of a sequence 3; and (b) protein which is derived from the protein of the sequence 3 and is obtained by processing the protein described in (a) by substituting and / or deleting and / or adding one or multiple amino acid residues related to plant resistance. GhCAT1 protein provided by the invention is protein described as (c) or (d): (c) protein formed by 85th to 576th amino acid residues from N end of a sequence 4; and (d) protein formed by 85th to 236th amino acid residues from N end of a sequence 3; and (b) protein which is derived from the protein of the sequence 4 and is obtained by processing the protein described in (a) by substituting and / or deleting and / or adding one or multiple amino acid residues related to plant resistance. According to the method, SOD1 gene and CAT1 gene of cotton are cloned, also the chloroplast signal peptide sequence of arabidopis thaliana THI1 gene is utilized for constructing chimeric gene, and the chimeric gene is overexpressed in cotton, so that the transgenic cotton with high salt resistance, drought resistance and disease resistance is cultivated. The technical scheme provided by the invention has important meaning on plant stress resistance, disease resistance and breeding.

Description

technical field [0001] The present invention relates to a method for cultivating resistant plants and its special protein and gene, in particular to a method of salt-resistant, drought-resistant and disease-resistant plant and its special protein and gene. Background technique [0002] Cotton is an important economic crop. In my country, the area of ​​cotton planted all year round is about 6.5 million hectares. [0003] Cotton is a crop with certain stress resistance, but as the frequency and degree of soil drought, salinization, and low-temperature freezing damage increase, its own stress resistance is no longer enough to resist, resulting in a significant decline in yield and quality . [0004] At the same time, cotton is a plant that is susceptible to the harm of pathogenic bacteria, and the yield and quality of cotton are greatly reduced due to diseases every year. Every year, the harm area of ​​verticillium wilt alone reaches 2 million hectares in my country, which is...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/02C12N15/53C12N9/08C12N15/62C12N15/63C12N5/10C12N1/21C12N15/82C12N15/84A01H5/00
CPCC07K2319/02C12N9/0065C12N9/0089C12N15/8273C12N15/8282C12Y111/01006C12Y115/01001
Inventor 吴家和庞金环夏桂先田颖川
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI