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Application of Leymus chinensis chitinase lcchi2 gene in improving plant cold tolerance

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

Active Publication Date: 2018-11-27
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 chitinase lcchi2 gene in improving plant cold tolerance
  • Application of Leymus chinensis chitinase lcchi2 gene in improving plant cold tolerance
  • Application of Leymus chinensis chitinase lcchi2 gene in improving plant cold tolerance

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

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

[0070] (1) RNA extraction: Leymus chinensis at the three-leaf stage was selected as the experimental material, and it was subjected to 100mM Na 2 CO 3 After treatment, take Leymus chinensis leaves in a mortar and grind them into powder with liquid nitrogen; extract total RNA with RNAiso at room temperature; use microspectrophotometer and agarose gel electrophoresis to detect the quality and purity of total RNA.

[0071] (2) Obtaining the 3'end fragment of LcChi2 gene: Use the SMARTTM RACE cDNA Amplification Kit of Clontech to obtain the 3'end fragment of the Leymus chinensis chitinase LcChi2 gene; according to the esterase gene EST (CD808914) sequence reported on Genbank Information Design of Leymus chinensis chitinase LcChi2 gene 3′-RACE primer, the primer sequence is as follows:

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

[0073] 5′-CCGCCGCCAACACCTTCC-3′

[0074] Reverse primer: 5'-GGCCACGCGTCGACTAGTACTTT...

Embodiment 2

[0083] Example 2: Sequence analysis of Leymus chinensis chitinase LcChi2 gene

[0084] Sequence analysis of the LcChi2 gene of Leymus chinensis chitinase: Perform multiple sequence alignment analysis of the LcChi2 gene of Leymus chinensis chitinase with homologous genes in other plants, and use DNAMAN software for sequence comparison. The same 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 all composed of methionine ( Met) initial.

[0085] Such as figure 1 As shown, the black boxes represent completely identical sequences, the gray boxes represent similar sequences, and the Roman numerals above the black lines represent possible motifs (conserved sequences in biological macromolecules such as DNA, proteins, etc., in the structure of trans-acting factors). Among them, the motif gener...

Embodiment 3

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

[0087] Construction of the phylogenetic tree of the chitinase LcChi2 gene of Leymus chinensis: The phylogenetic tree was constructed and evaluated by MEGA 3.1 and Clustal X programs. The construction of a phylogenetic tree can predict or analyze the possible functions 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, gene type and species such as figure 2 Shown:

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

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

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

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

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

[0093] BAD77932-CjCHI-IV: Cryptomeria fortunei class IV chitinase[Crypt...

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Abstract

The application of the Leymus chinensis LcChi2 gene in improving the cold resistance of plants relates to the field of plant molecular biology. The application method comprises the following steps: (1) After operably connecting the Leymus chinensis LcChi2 gene to the expression control sequence on the vector, a recombinant vector containing the nucleotide sequence shown in SEQ ID NO: 1 is formed; ( 2) transforming the host plant cells with the recombinant vector obtained in step (1), and obtaining transformed cells through screening; (3) cultivating the transformed cells obtained in step (2) into transgenic LcChi2 gene plants with cold tolerance and their progeny. In step (1), the vector is the plant expression vector pBI121 or the plant expression vector pCAMBIA3300. Experiments have confirmed that the seed germination rate of transgenic LcChi2 tobacco has been significantly improved in cold environment, and the overexpression of LcChi2 gene in dicotyledonous plant tobacco, monocotyledonous plant maize and rice has significantly improved the cold tolerance of plant seedlings, thus proving that overexpression Expression of LcChi2 gene significantly improves the cold tolerance of tobacco, corn, rice and other plants.

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 tolerance. Background technique [0002] Leymus chinensis Trin (Leymus chinensis Trin or Aneurolepidium chinense Trin), also known as Elymus vulgaris, is a perennial herb of the family Leymus genus. Leymus chinensis has a lot of leaves, is rich in nutrition, and has good palatability. All kinds of livestock like to eat all year round, and it has the reputation of "fine grain for livestock". Leymus chinensis has the advantages of salt and alkali tolerance, cold tolerance, drought tolerance, fungus resistance, and bacteria resistance, and can adapt to growth in plains, hillsides, and sandy loam soils. Leymus chinensis is very resistant to salt and alkali. It contains many resistance-related proteins in its body and is a good material for cloning resistance genes. [0003] Chitinase (chiti...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/56C12N15/82A01H5/00A01H6/46A01H6/82
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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