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LRR-RLK (leucine-rich repeats-receptor-like kinase) in arabidopsis thaliana and application thereof

A technology of protein kinase and Arabidopsis thaliana, applied in the field of genetic engineering, can solve the problems of large investment and low benefit, and achieve the effect of strong stress resistance and low degree of cell damage

Inactive Publication Date: 2015-11-11
WUHAN BINGGANG BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the improvement of saline-alkali land is a worldwide problem, and traditional measures require large investment and low benefits

Method used

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  • LRR-RLK (leucine-rich repeats-receptor-like kinase) in arabidopsis thaliana and application thereof
  • LRR-RLK (leucine-rich repeats-receptor-like kinase) in arabidopsis thaliana and application thereof
  • LRR-RLK (leucine-rich repeats-receptor-like kinase) in arabidopsis thaliana and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Embodiment one: Arabidopsis LRR-RLK protein kinase protein gene AtRLK clone

[0030] 1. Extraction of total RNA from Arabidopsis thaliana

[0031] All experimental drugs were purchased from Zhongke Ruitai Bioengineering Co., Ltd. The mortars, pipette tips, centrifuge tubes and other items used should be soaked in 1% DEPC water overnight, and then sterilized under high temperature and high pressure for later use.

[0032] (1) RNA extraction reference (TRIZOL TM KitRNA Extraction Reagent Manual)

[0033] (2) Take 50 mg of fresh plant tissue samples and grind them with liquid nitrogen;

[0034] (3) Add 1ml TRIZOL and place at room temperature (20-25°C, the same below) for 10min;

[0035] (4) Add 200 μl of chloroform, shake vigorously for 60 seconds, and place at room temperature for 5 minutes;

[0036] (5) Centrifuge at 12,000 rpm for 10 min (4°C), take 500 μl of supernatant into a new PE tube, add 1 ml of absolute ethanol, mix well, and place at -20°C for 20 min;

[...

Embodiment 2

[0053] Embodiment two: Utilize pCAMBIA1301 vector construction CaMV35S - AtRLK fusion gene

[0054] (1) Extract vector pCAMBIA1301 plasmid (purchased from CAMBIA Company) from Escherichia coli, use Nco I / BstE After II double enzyme digestion, the target vector fragment is recovered.

[0055] (2) to the reclaimed of embodiment 1 AtRIPK The CDS fragment of the gene is used Nco I / BstE II double enzyme digestion, and recover the digested fragments (same as in Example 1) by agarose gel electrophoresis.

[0056] (3) The above two fragments were ligated overnight at 16°C under the catalysis of ligase to complete the pCAMBIA1301 carrier CaMV35S - AtRLK Fusion gene construction.

[0057] Connection system:

[0058] AtRLK Gene CDS fragment (50ng / μl): 2μl;

[0059] pCAMBIA1301 vector (50ng / μl): 3μl;

[0060] SolutionI ligase 5 μl;

[0061] (4) Transform Escherichia coli DH5α competent cells with the ligation mixture, the specific method is as follows:

[0062] Pre...

Embodiment 3

[0071] Example 3: Preparation of transgenic Arabidopsis plants

[0072] (1) constructed with embodiment two CaMV35S - AtRLK The fusion gene was transformed into Arabidopsis thaliana, and the obtained seeds were subjected to 50mgl -1 For hygromycin resistance screening, normal growing plants were transferred to nutrient soil for culture.

[0073] (2) Real-time quantitative RT-PCR detection of transgenic plants: the transgenic Arabidopsis thaliana material identified by PCR in Example 2 is multiplied, after 50mgl -1 Hygromycin resistance screened for T 3 Homozygous transgenic lines were generated, wild-type and transgenic Arabidopsis seedlings grown for one week were respectively obtained, and total RNA from plant tissues was extracted according to the method in Example 1 and reverse-transcribed into mRNA. SYBRGreenRealtimePCRMasterMixplus (TOYUBO, QPK-212) kit was used for qRT-PCR detection, and the operation was completed according to the kit instructions.

[0074] Detec...

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Abstract

The invention relates to salt tolerance related LRR-RLK (leucine-rich repeats-receptor-like kinase) in arabidopsis thaliana and an application thereof to cultivation of salt tolerant transgenic plants. The amino acid sequence of LRR-RLK of a protein is shown in SEQ ID No.2 and a coded nucleotide sequence is shown in SEQ ID No.1. The protein related to salt tolerance of plants and a coding gene thereof, which are provided by the invention, have important significance in improving and reinforcing the stress resistance of arabidopsis thaliana and then accelerating the stress resistant molecular breeding process and have realistic application values in agricultural production in China.

Description

technical field [0001] The invention relates to an Arabidopsis thaliana LRR-RLK protein kinase and its application in cultivating salt-tolerant plants, belonging to the technical field of genetic engineering. Background technique [0002] The problem of soil salinization (Soilsalinization) is widespread in the world, especially in arid and semi-arid areas, where the problem is more serious. Salt stress has become one of the important abiotic stresses affecting global crop yields. According to incomplete statistics from UNESCO, the global saline-alkali land area has reached 950 million hm 2 , accounting for about 10% of the total land area of ​​the earth, of which China is 99.13 million hm 2 . The main characteristics of soil salinization in my country are large area, wide distribution, many types, and the salinization and secondary salinization are increasing every year. Soil salinization has become the main factor restricting the sustainable development of agriculture in...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/12C12N15/54C12N15/82C12N15/66A01H5/00
CPCC12N9/1205
Inventor 王宇峰杨冰范艺翔郑媛坤束礼平束礼伟何银竹黄刚
Owner WUHAN BINGGANG BIOTECH CO LTD
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