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Cotton salt-tolerant gene GarCIPK for improving plant salt tolerance

A technology of salt tolerance and cotton, applied in the field of biological genetic engineering, can solve the problem of less research on cotton, and achieve the effect of improving salt tolerance

Inactive Publication Date: 2012-08-08
JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, some members of the CBL-CIPK signaling system have been identified, and the function of this signaling network in plant response to adversity stress has been analyzed in the study of some model plants, but there are few related studies in cotton

Method used

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  • Cotton salt-tolerant gene GarCIPK for improving plant salt tolerance
  • Cotton salt-tolerant gene GarCIPK for improving plant salt tolerance
  • Cotton salt-tolerant gene GarCIPK for improving plant salt tolerance

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Implementation example 1, GarCIPK Gene acquisition

[0020] 1.1 Extraction of RNA

[0021] extract RNA

[0022] (1) Take 0.5g of fresh cotton tissue, add 0.1g of cross-linked polyvinylpyrrolidone (PVPP), fully grind to powder in liquid nitrogen, quickly transfer the frozen powder into a 10ml centrifuge tube, add 5ml of CTAB extract With 500 μL of 0.1M Tris-HCl of pH 8.0, bathe in water at 65°C for 20 minutes, and mix well by inverting halfway;

[0023] (2) Add an equal volume of chloroform to mix thoroughly, and let stand in an ice bath for 10 minutes;

[0024] (3) Centrifuge at 10,000 rpm for 20 minutes at 4°C. Divide into four 1.5ml centrifuge tubes;

[0025] (4) Aspirate the supernatant, add 1 / 3 volume of 8M LiCl, mix well, and incubate at -70°C for 30min or -20°C overnight;

[0026] (5) Centrifuge at 10,000 rpm for 20 minutes at 4°C. Discard the supernatant, wash twice with 70% ethanol, dry the precipitate and dissolve it in 30 μL DEPC water;

[0027] (6)...

Embodiment 2

[0076] Embodiment 2, the construction of plant expression vector

[0077] 2.1 Construction of pCAMBIA2301-CaMV35S-GarCIPK plant expression vector

[0078] Plant expression vector pCAMBIA2301-CaMV35S (purchased from Biovector Science Lab) plasmid, select BamHI and KnpⅠ to digest pCAMBIA2301 and the target gene fragment respectively, recover the large vector fragment and the target gene fragment, connect with T4 ligase and transform Escherichia coli DH5α to sense state cells, and the plant expression vector with the target gene can be obtained after the recombinant is identified.

[0079] Enzyme digestion of pCAMBIA2301 plasmid and target gene fragment

[0080] The plasmid double enzyme digestion system is as follows:

[0081] 10×K buffer 5μL

[0082] 2301 plasmid 25μL

[0083] BamHI 0.5 μL

[0084] KnpI 0.5 μL

[0085] sterile ddH 2 O 19 μL

[0086] Enzyme digestion at 30°C for 5h. After the double enzyme digestion, the agaro...

Embodiment 3

[0111] Embodiment 3, preparation and transformation of Agrobacterium competent

[0112] 3.1 Preparation of Competent Agrobacterium EHA105

[0113] (1) Pick a single colony of EHA105, inoculate it in 5ml LB liquid medium, and culture overnight at 28°C with shaking at 200rpm until the OD600 value is 0.4;

[0114] (2) Inoculate in 400-500ml LB medium (in a 1L Erlenmeyer flask) at a ratio of 1:100, shake the bacteria until the OD600 is 0.6-0.8, and bathe in ice for 10 minutes;

[0115] (3) Collect the bacterial liquid in a pre-cooled 50ml centrifuge tube, centrifuge at 5000rpm at 4°C for 5min;

[0116] (4) Discard the supernatant, and use 10ml pre-cooled 0.1MCaCl for precipitation 2 Fully suspend, 4°C, 5000rpm, centrifuge for 5min;

[0117] (5) Add 4ml (depending on the number of bacteria) to pre-cool the solution containing 15% 0.1mol / L CaCl2, gently suspend the cells, and place them on ice for a few minutes to form a competent cell suspension;

[0118] (6) Divide into 100μL-...

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Abstract

The invention discloses a cotton salt-tolerant gene, i.e. Gossypium wild dry land cotton CIPK-like protein phosphokinase GarCIPK. The gene has the sequence of SEQ ID No.1 in a sequence list. A cotton stress tolerance related gene is separated by an electronic cloning and reverse transcription-polymerase chain reaction (RT-PCR) technology, and function verification is performed by transferring gene into tobacco and shows that the salt tolerance of a transgenic plant is obviously improved.

Description

technical field [0001] The invention belongs to the technical field of biogenetic engineering, and relates to a cotton for improving the salt tolerance of plants GarCIPK Gene. Background technique [0002] Soil salinization is a global resource and environmental problem and an ecological problem. According to statistics, there are about 1 billion hectares of salinized land in the world, accounting for about 10% of the world's total land area, while my country has nearly 100 million hectares of salinized land. With the development of economy, the industrial The aggravation of pollution, irrational irrigation and improper application of chemical fertilizers have caused the area of ​​secondary salinized soil to expand year by year, seriously hindering the sustainable development of agriculture. Improvement and utilization, comprehensive management of saline soil, and cultivation of new salt-tolerant varieties to improve plant salt tolerance have become major issues in the dev...

Claims

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Application Information

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IPC IPC(8): C12N15/54C12N9/12C12N15/82
Inventor 沈新莲冯娟刘章伟徐鹏张香桂
Owner JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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