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Gene of wintersweet late embryogenesis abundant protein (LEA) and low-temperature resistant application thereof

A technology of embryo development and enrichment, applied in the field of molecular biology and genetic engineering, to achieve the effect of improving the ability to resist low temperature

Inactive Publication Date: 2011-04-13
JILIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] In many studies, there is no relevant report about LEA in the multi-resistant plant wintersweet, and wintersweet [Chimonanthus praecox], as a species with multi-resistant characteristics, has a relatively strong adaptability to the external environment, and wintersweet is resistant to Drought, cold resistance, less pests and diseases, there must be multiple resistance genes in wintersweet

Method used

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  • Gene of wintersweet late embryogenesis abundant protein (LEA) and low-temperature resistant application thereof
  • Gene of wintersweet late embryogenesis abundant protein (LEA) and low-temperature resistant application thereof
  • Gene of wintersweet late embryogenesis abundant protein (LEA) and low-temperature resistant application thereof

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Experimental program
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Effect test

Embodiment 1

[0034] Example 1: Cloning of the CpLEA3 gene

[0035] 1. Extract RNA:

[0036] Get 500mg wintersweet corolla and use RNAiso Reagent to extract wintersweet flower total RNA.

[0037] 2. Construction of cDNA library:

[0038] The total RNA was taken, and the cDNA library of Wintersweet Flower was synthesized according to the SMARTTM cDNA Library Construction Kit, and 1000 library clones were randomly selected for sequencing and bioinformatics analysis to obtain the sequence of Wintersweet Flower ESTs.

[0039]3. Cloning of CpLEA3 gene fragment:

[0040] According to the cDNA sequence encoding CpLEA3 found in the ESTs sequence of Wintersweet flower, the upstream and downstream primers were designed, and the PCR amplification was carried out with the cDNA library of Wintersweet flower as a template.

[0041] Specific primers are as follows:

[0042] CpLEA3-P1: 5'-CGGGATCCATGGCTCGCTCTCTGTTG-3', bases in italics are BamH I restriction sites;

[0043] CpLEA3-P2: 5'-CGAGCTCGTGGTT...

Embodiment 2

[0049] Example 2: High expression of CpLEA3 in bacterial strains

[0050] 1. Construction of prokaryotic expression vector:

[0051] In order to demonstrate the coding function of the CpLEA3 gene, the CpLEA3 gene was connected between the Sac I and BamH I sites of pET-32a(+), and transformed into Escherichia coli BL21 to obtain high-level expression.

[0052] Design and synthesize a pair of specific primers:

[0053] 5'-CGGGATCCATGGCTCGCTCTCTGTTG-3', bases in italics are BamH I restriction sites;

[0054] 5'-CGAGCTCGTGGTTACGGAATTTCTGGG-3', bases in italics are Sac I restriction sites.

[0055] According to the conventional experimental procedure of molecular cloning, the coding sequence of the gene with specific restriction sites at both ends was amplified by PCR method, and the gene was connected with the vector pMD18-T (purchased from Shanghai Dingguo Biotechnology Co., Ltd.) to verify that it was correct. Take the pMD18::CpLEA3 plasmid and the pET-32a(+) plasmid (purchas...

Embodiment 3

[0058] Example 3: Determination of cell viability of bacterial strains after low temperature stress

[0059] Take a single colony of the positive clone and the empty vector strain, respectively, in 5 mL of LB liquid medium containing 100 μg / mL Amp, and culture it at 37 °C with shaking at 200 r / min until OD 600 0.5, add IPTG to a final concentration of 1mmol / L, and continue to cultivate for 3h. Divided into 2 parts respectively, the first part was diluted 10000 times, spread on LB medium containing 100 μg / mL Amp, and cultured overnight at 37°C; the second part was sucked 1mL and subjected to low temperature stress treatment at -20°C for 24h, 48h and 72h After treatment, they were diluted 10,000 times and spread on LB medium containing 100 μg / mL Amp, and cultured overnight at 37°C. Count the number of colonies on the next day (such as Figure 4 ), the survival rate was calculated by the ratio of the number of colonies grown after stress to the control group without stress (suc...

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Abstract

The invention relates to a gene of wintersweet late embryogenesis abundant protein (LEA) and low-temperature resistant application thereof, belonging to the field of molecular biology and gene engineering. The invention provides a gene of wintersweet LEA and protein and also provides an expression vector and a host cell corresponding to the gene. The invention has the advantage that the application of the gene of the wintersweet LEA can improve the low-temperature resistance of a strain.

Description

technical field [0001] The invention belongs to the field of molecular biology and genetic engineering, and specifically relates to a gene cloning and application of abundant protein in late embryonic development of wintersweet. Background technique [0002] Drought, salinity and cold temperatures can all cause water deficits in plants, during which plant cells accumulate a series of proteins that protect cells from dehydration. In addition to causing metabolic changes and the accumulation of low-molecular-weight protective compounds, environmental stress can activate transcription of many genes in plants and lead to the accumulation of new proteins in plant vegetative tissues, especially the research on plant drought and low-temperature-induced proteins has attracted widespread attention. , among them, late embryogenesis abundant protein (late embryogenesis abundant protein, LEA) refers to a series of proteins accumulated in large quantities in seeds in the late embryogenes...

Claims

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Application Information

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IPC IPC(8): C12N15/29C12N15/70C12N1/21C07K14/415C12R1/19
Inventor 张世宏谢丽霞陈宣明高文阳晓红张莉弘刘金亮
Owner JILIN UNIV
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