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Application of Leymus chinensis Trin chitinase (LcChi2) gene in improving cold resistance of plants

A chitinase and cold tolerance technology is applied in the application field of Leymus chinensis chitinase LcChi2 gene in improving the cold tolerance of plants, and can solve the problems that the research on the Leymus chinensis chitinase LcChi2 gene has not been reported, etc. The method is simple and effective, has good application prospects, and improves the effect of plant cold tolerance

Active Publication Date: 2015-10-14
JILIN ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] At present, there is no report on the effect of L. chinensis chitinase LcChi2 gene on improving plant cold tolerance.

Method used

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  • Application of Leymus chinensis Trin chitinase (LcChi2) gene in improving cold resistance of plants
  • Application of Leymus chinensis Trin chitinase (LcChi2) gene in improving cold resistance of plants
  • Application of Leymus chinensis Trin chitinase (LcChi2) gene in improving cold resistance of plants

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0069] Example 1: Cloning of Leymus chinensis LcChi2 gene

[0070] (1) Extraction of RNA: Select Leymus chinensis at the three-leaf stage as the experimental material, after 100mM Na 2 CO 3 After treatment, Leymus chinensis leaves were taken in a mortar and ground into powder with liquid nitrogen; total RNA was extracted by RNAiso at room temperature; the quality and purity of total RNA were detected by micro-spectrophotometer and agarose gel electrophoresis.

[0071] (2) Obtaining the 3′ end fragment of the LcChi2 gene: using the SMARTTM RACE cDNA Amplification Kit kit from Clontech Company, the 3′ end fragment of the LcChi2 gene of Leymus chinensis was obtained; according to the esterase gene EST (CD808914) reported on Genbank Sequence information Design 3'-RACE primers of Leymus chinensis LcChi2 gene, the primer sequence is as follows:

[0072] Forward primer: 5′-CCGACCAGTTCCAATGGGGCT-3′

[0073] 5′-CCGCCGCCAACACCTTCC-3′

[0074] Reverse primer: 5′-GGCCACGCGTC...

Embodiment 2

[0083] Example 2: Sequence Analysis of Leymus chinensis LcChi2 Gene

[0084] Sequence analysis of LcChi2 gene of Leymus chinensis LcChi2: multiple sequence alignment analysis of chitinase LcChi2 gene of Leymus chinensis and homologous genes in other plants, using DNAMAN software for sequence comparison, homologous genes in other plants and other plants The accession numbers of the source genes in GenBank are: Hordeum vulgare (CAA55344), Triticum aestivum (BAB82471), Secale cereale (AAG53610) and Capsicum annuum (AAC36359), these four homologous gene sequences are composed of methionine ( Met) started.

[0085] like figure 1 As shown, the black box represents a completely consistent sequence, the gray box represents a similar sequence, and the Roman numerals above the black line represent possible motifs (conserved sequences in biological macromolecules such as DNA and proteins, in the structure of trans-acting factors Among them, the motif generally refers to the basic struc...

Embodiment 3

[0086] Example 3: Construction of phylogenetic tree of Leymus chinensis LcChi2 gene

[0087] Construction of phylogenetic tree of chitinase LcChi2 gene of Leymus chinensis: MEGA 3.1 and Clustal X programs were used to construct phylogenetic tree and evaluate phylogenetic tree. The construction of the phylogenetic tree can predict or analyze the possible function of the target gene, for example, the target gene is likely to be the same or similar to the known gene closest to it. Accession number, genotype and species such as figure 2 Shown:

[0088] CAA55344-HvCHI-II: Barley chitinase [Hordeum vulgare subsp.vulgare];

[0089] ACV20870-LcChi2: Leymus chinensis [Leymus chinensis];

[0090]BAB82471-TaCHI-II: Wheat chitinase 1 [Triticum aestivum];

[0091] AAG53610-ScCHI-II: Rye 24.8kDa class II endochitinase-antifreeze protein precursor [Secale cereale];

[0092] AAC36359-CaCHI-II: Capsicum chitinase class II [Capsicum annuum];

[0093] BAD77932-CjCHI-IV: Cryptomeria class ...

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Abstract

The invention relates to an application of Leymus chinensis Trin chitinase (LcChi2) gene in improving cold resistance of plants, relating to the field of the plant molecular biology. The application method comprises the following steps: (1) connecting the LcChi2 gene on an expression regulation sequence on a carrier in an operationable manner, so as to form a recombinant vector containing the nucleotide sequence shown in SEQ ID NO:1; (2) transforming host cells of the recombinant vector obtained in the step (1), and screening to obtain transformed cells; and (3) culturing the transformed cells obtained in the step (2) to obtain a LcChi2 gene transfer plant with cold resistance and the offspring of the LcChi2 gene transfer plant. In the step (1), the vector is plant expression vector pBI121 or plant expression vector pCAMBIA3300. The test proves that the seed germination rate of LcChi2 gene transfer tobacco in the cold environment is obviously improved, overexpression LcChi2 genes of dicotyledon tobacco and monocotyledon corn and rice obviously improve the cold resistance of the plants during the seedling stage, so that the result shows that the overexpression LcChi2 gene obviously improves the cold resistance of plants, including tobacco, corns, rice and the like.

Description

technical field [0001] The invention relates to the technical field of plant molecular biology, in particular to the application of a Leymus chinensis chitinase LcChi2 gene in improving plant cold resistance. Background technique [0002] Leymus chinensis Trin (Leymus chinensis Trin or Aneurolepidium chinense Trin), also known as Alkali grass, is a perennial herbaceous plant belonging to the family Gramineae. Leymus chinensis has many leaves, rich nutrition and good palatability. All kinds of livestock like to eat it all year round. It has the reputation of "fine grain for livestock". Leymus chinensis has the advantages of salt-alkali resistance, cold resistance, drought resistance, anti-fungal, anti-bacterial, etc., and can adapt to growth in plains, hillsides, and sandy loam. Leymus chinensis is very resistant to salt and alkali, and contains many resistance-related proteins in its body, which is a good material for cloning stress-resistant genes. [0003] Chitinase (chi...

Claims

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Application Information

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IPC IPC(8): C12N15/56C12N15/82A01H5/00
Inventor 刘相国郝东云尹悦佳柳青韩四平李楠林秀峰刘艳芝金学博刘洋
Owner JILIN ACAD OF AGRI SCI
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