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y associated with stress resistance of fennel beans 2 k 4 dehydrin mry 2 k 4 and its coding genes and applications

A dehydrin protein, Y2K4 technology, applied in the Y2K4 type dehydrin protein MrY2K4 and its coding gene and its application field, can solve the unseen reports on the cloning of the stress-resistant dehydrin protein gene and the identification of the stress-resistance function of lentil beans, etc. problem, to achieve the effect of improving drought resistance

Active Publication Date: 2022-02-01
QINGHAI UNIV FOR NATITIES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] There are no reports on the cloning of the stress-resistant dehydrin protein gene of the YnKn type and the identification of the stress-resistance function in fenugreek

Method used

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  • y associated with stress resistance of fennel beans <sub>2</sub> k <sub>4</sub> dehydrin mry <sub>2</sub> k <sub>4</sub> and its coding genes and applications
  • y associated with stress resistance of fennel beans <sub>2</sub> k <sub>4</sub> dehydrin mry <sub>2</sub> k <sub>4</sub> and its coding genes and applications
  • y associated with stress resistance of fennel beans <sub>2</sub> k <sub>4</sub> dehydrin mry <sub>2</sub> k <sub>4</sub> and its coding genes and applications

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1 clover bean dehydrin protein MrY 2 K 4 gene cloning

[0030] 1. Low-temperature stress treatment and transcriptome sequencing of fenugreek seedlings

[0031] (1) Low temperature stress treatment of lentil beans

[0032] The linalo seeds were treated with concentrated sulfuric acid for 10 minutes, during which they were constantly stirred with a glass rod, and then washed several times with distilled water to remove sulfuric acid. The above-mentioned treated seeds were placed in a petri dish covered with multiple layers of wet filter paper, and germinated under the conditions of 21°C and 16h / 8h photoperiod; after 3 days, the germinated seedlings were transplanted in vermiculite: nutrient soil (3:1) Continue to cultivate under the above conditions in the mixed medium, and irrigate once a week with nutrient solution containing 1 / 2MS; after 3 weeks, transfer the seedlings to an intelligent artificial climate incubator, and treat them at a low temperature of 4°C...

Embodiment 2

[0074] Example 2 The dehydrin gene MrY related to the stress resistance of lentils 2 K 4 Analysis of the expression characteristics of

[0075] 1. Abiotic stress and ABA treatment of flat clover bean seedlings

[0076] (1) By the method in embodiment 1, the seeds of the flat clover bean are treated with concentrated sulfuric acid, germinated, transplanted and grown to 3 weeks later, carry out abiotic stress and ABA treatment, get the whole plant seedling at different treatment time points, after sampling Quick-frozen in liquid nitrogen and stored in a -80°C refrigerator for extraction of total RNA.

[0077] (2) Drought treatment: Remove the seedlings from the culture substrate, wash the root substrate with distilled water, absorb excess water with absorbent paper, place it in a petri dish, and dehydrate it naturally at room temperature. 6h and 8h, take the whole seedlings.

[0078] (3) NaCl stress treatment: water from the bottom of the flowerpot with 150mmol / L NaCl aqueou...

Embodiment 3

[0093] Example 3 The dehydrin gene MrY related to the stress resistance of lentil beans 2 K 4 Prokaryotic expression of

[0094] 1. Primer design for prokaryotic expression:

[0095] Primers P3 and P4 were designed according to the sequencing results to amplify MrY 2 K 4 DNA sequence of the coding region of the gene, and two restriction sites (underlined), BamHI and SacI, were introduced at the 5'-end and 3'-end of the DNA sequence, respectively.

[0096] P3: 5'-CGC GGATCC ATGTCTCAATATCAACAAAGTCAT-3' (SEQ ID NO: 7)

[0097] P4: 5'-CTGGACCACGTACTAGACAGTAG GAGCTC GCG-3' (SEQ ID NO: 8)

[0098] 2. Extract the recombinant plasmid and expression vector pET-30a(+) plasmid (Novagen product) with the plasmid mini-extraction kit (Shanghai Sangon product).

[0099] 3. MrY 2 K 4 PCR amplification and cloning of gene coding region sequence:

[0100] The PCR reaction system consisted of 25 μL 2×LA Buffer I, 4 μL dNTP (25Mm), 1 μL P3 (10 μM), 1 μL P4 (10 μM), 0.14 μL LA TM Taq...

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Abstract

The invention provides a kind of Y related to stress resistance of lentil beans 2 K 4 dehydrin MrY 2 K 4 And its coding gene and its application. The present invention clones and isolates a new YnKn-type dehydrin protein and its coding gene MrY from lentil beans by RT-PCR technology 2 K 4 , and use prokaryotic expression to express MrY 2 K 4 After the gene coding region is connected to the E. coli expression vector, the prokaryotic expression vector is transferred into the E. coli expression strain, and MrY is realized after induction with IPTG 2 K 4 Protein overexpression in E. coli, the results showed that MrY 2 K 4 The overexpression of the protein has a protective effect on Escherichia coli under adversity stress, therefore, the gene can be used to improve the salt resistance and heat resistance of plants and microorganisms through biotechnology methods.

Description

technical field [0001] The invention belongs to the technical field of biogenetic engineering, and in particular relates to a Y 2 K 4 dehydrin MrY 2 K 4 And its coding gene and its application. Background technique [0002] Medicago ruthenica (L.), also known as alfalfa, lentils, wild alfalfa, etc., is an annual or perennial leguminous plant of the genus Medicago. Flat clover beans have wide ecological adaptability, and are distributed in northern and southwestern my country, Siberia, and Mongolia, and mostly grow on hillside grasslands. It has been found through research that flat clover beans have strong cold resistance, salt tolerance and drought resistance, and are the only leguminous alfalfa plants that can adapt to drought, severe cold on the plateau and poor soil. [0003] Late embryogenesis abundant protein (LEA) was first discovered in seeds, and subsequent studies have shown that it is widely distributed in various tissues and organs of algae, yeast, nematodes...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/415C12N15/29C12N15/70C12N1/21C12N15/82C12R1/19
CPCC07K14/415C12N15/70C12N15/8273C12N15/8271
Inventor 沈迎芳王海庆马超张湑泽
Owner QINGHAI UNIV FOR NATITIES
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