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Cold resistance correlated protein from leymus chinensis, encoding gene thereof and application thereof

A coding and gene technology, applied in the field of genetic engineering, can solve problems such as complex response mechanism, hinder large-scale application, and difficult to specifically enhance cold resistance, and achieve broad application prospects, improve cold resistance, and high application value.

Active Publication Date: 2016-01-13
INST OF BOTANY CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although people have done a lot of research on the process mechanism of plant response to low temperature stress for many years, many important issues in plant response to low temperature still need to be further explored due to the complexity of the response mechanism of plants to external environmental changes.
At the same time, due to the complexity of many intercrossing pathways in the stress resistance mechanism of plants, it is difficult to obtain varieties with enhanced cold resistance using traditional cross breeding methods.
In addition, the screening of high-quality cold-resistant genes has also become an important factor hindering the large-scale application of plant low-temperature-resistant genetic engineering.

Method used

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  • Cold resistance correlated protein from leymus chinensis, encoding gene thereof and application thereof
  • Cold resistance correlated protein from leymus chinensis, encoding gene thereof and application thereof
  • Cold resistance correlated protein from leymus chinensis, encoding gene thereof and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0070] Example 1. Obtaining the full-length cDNA sequence and genome sequence of the cold resistance-related gene Lc3919 of Leymus chinensis

[0071] 1. Cloning of the 5' end sequence of Lc3919, a gene related to cold resistance of Leymus chinensis

[0072] 1. Plant material processing and total RNA extraction

[0073] Leymus chinensis (Trin.) Tzvel.) variety Zhongke 2 (recorded in "XXLi, SLHou, QGao, et al. LcSAIN1, novell salt-induced gene from sheepgrass, conferssalt stress tolerance in transgenic Arabidopsis andrice. One article, the public can obtain from the Institute of Botany, Chinese Academy of Sciences) seedlings as materials, extract total RNA after low temperature stress (4°C) treatment for 12 hours, and carry out 1% agarose gel electrophoresis detection, the results are as follows figure 1 shown. The extracted RNA has two obvious electrophoresis bands, which are 28SRNA and 18SRNA from top to bottom, indicating that the total RNA with higher purity and integrity ...

Embodiment 2

[0092] Example 2. Expression pattern and tissue specificity analysis of Lc3919 gene under stress conditions

[0093] The seedlings of Leymus chinensis variety Zhongke No.2, which had grown normally for 5 weeks, were subjected to low temperature stress (4°C). The stress treatment time was 0, 1, 2, 4, 8, 12, 24, 48 hours, respectively. The total RNA of Leymus chinensis seedlings treated above was extracted, and the expression pattern of Lc3919 gene under low temperature stress was analyzed by qRT-PCR method. Wherein the amplified Lc3919 gene primer sequence is 5'-CATCTTCTTCCTCAGCGCAC-3' (position 511-530 of sequence 2) and 5'-GACTCATCCTCCACATCGCCG-3' (reverse complementary sequence of position 604-623 of sequence 2); Actin was used as an internal reference gene, and the primer sequences for amplifying the internal reference Actin were 5'-GTGCTTTCCCCTCTATGCAAGTGGT-3' and 5'-CTGTTCTTGGCAGTCTCCAGCTC-3'; two-step PCR amplification standard procedure: 95°C for 10s; 95°C for 5s, 60°C...

Embodiment 3

[0097] Example 3, the subcellular localization of the Lc3919 gene

[0098] The Lc3919 gene amplified in Example 1 above (positions 1-907 of sequence 2) was connected to the pMDC45 vector containing GFP through a recombinant LR reaction (recorded in "Mark Curtis, Ueli Grossniklaus, et al. Agateway cloning vectorset for high-throughput functional analysis of genes in planta. (2003). PlantPhysiology 133:462-469." article, the public can obtain from the Institute of Botany, Chinese Academy of Sciences) between the recombination sites attR1 and attR2, the resulting recombinant expression vector was named pMDC45-Lc3919. The structure of the recombinant expression vector pMDC45-Lc3919 is described as: a recombinant plasmid formed by inserting the DNA fragment shown in nucleotides 1-907 of Sequence 2 in the sequence listing between the recombination sites attR1 and attR2 of the pMDC45 vector.

[0099] The recombinant expression vector pMDC45-Lc3919 was introduced into Agrobacterium tu...

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Abstract

The present invention discloses a cold resistance correlated protein from leymus chinensis, an encoding gene thereof and an application thereof. The protein provided by the invention is (a) or (b): (a) a protein with an amino acid sequence as shown in a sequence 1 in a sequence list; (b) a protein obtained by substituting and / or deleting and / or adding one or more amino acid residues of the amino acid sequence as shown in the sequence 1 and related with plant cold resistance. The protein related with the plant cold resistance and the encoding gene Lc3919 of the protein provided by the invention are induced at a low temperature; a transgenic plant of the protein has relatively high cold resistance; according to the experiments, a wild type arabidopsis plant subjected to overnight cold stress at (-)8 DEG C only has a survival rate of 23.33% while the survival rate of the Lc3919 transgenic plant can be increased to 46.29% which is twice that of the survival rate of wild type arabidopsis. The Lc3919 gene can be used as a candidate gene of plant cold resistance genetic engineering for improving cold resistance of a plant.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and relates to a protein derived from Leymus chinensis and related to cold resistance, its coding gene and application. Background technique [0002] In spring, autumn and winter, plants in cold regions are often in sub-zero environments, and thus need a series of molecular and physiological adaptations to reduce the damage caused by freezing damage to themselves. Facing the frequent alternation of warm winters and extreme severe weather caused by global temperature rise, the loss of crops due to low temperature and freezing damage is as high as hundreds of billions of yuan every year. Therefore, it is necessary to strengthen the mining of cold-resistant genes of plants, especially those plants (such as crops, pastures, etc.) Grass cold resistance is a major problem and challenge that my country and the world's agriculture and animal husbandry are currently facing. [0003] The characteristic ...

Claims

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Application Information

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IPC IPC(8): C07K14/415C12N15/29C12N15/82C12N1/15C12N1/19C12N1/21C12N15/11
Inventor 刘公社高琼程丽琴李晓霞赵品苍齐冬梅陈双燕
Owner INST OF BOTANY CHINESE ACAD OF SCI
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