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Millet silea14 gene and its application

A millet, gene technology, applied in the application, genetic engineering, plant genetic improvement and other directions, to achieve the effect of improving plant stress resistance and increasing plant stress resistance

Inactive Publication Date: 2016-10-05
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, there are no reports about other 5C subfamily LEA genes improving the stress resistance of transgenic plants

Method used

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  • Millet silea14 gene and its application
  • Millet silea14 gene and its application
  • Millet silea14 gene and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1 Cloning of plant resistance stress-related gene SiLEA14

[0038] Select the immature seeds of millet on the 5th, 7th and 12th day after pollination. After the materials are mixed, total RNA is extracted, mRNA is enriched by the magnetic bead method (the kit is purchased from Promega Company), and the cDNA Synthesis Kit of Stratagene Company is used for reverse transcription synthesis. cDNA, after the double-stranded cDNA was filled in, the EcoRI linker was connected, and it was constructed on the λZAPII vector, and ZAP-cDNA GigapackIII Gold Cloning Kit (Stratagene Company) was used for in vitro packaging. After packaging, SM solution and chloroform were added, and after centrifugation, the supernatant was the constructed library. Take 1 μl of library supernatant for titer determination, the results show that the capacity of the constructed library is 2×10 7 pfu / ml. After the original library is constructed, it is amplified once and set aside.

[0039] The s...

Embodiment 2

[0041] Example 2 Construction and Agrobacterium Transformation of Expression Vectors for Expressing SiLEA14 Gene in Arabidopsis

[0042] The expression cassette is obtained from the Super promoter of Agrobacterium tumefaciens (that is, the mas promoter is fused with 3 ocs enhancers and 1 mas enhancer, see Leisner SM, Gelvin SB (1988) Structure of the octopinesynthse upstream activator sequence. PNAS85, 2553-2557) and the 3' transcription termination region from the nopaline synthase (nos) gene (Depicker A, Stachel S., Dhaese P, Zam bryski P, Goodman HM (1982). Nopaline synthase: transcript mapping and DNA sequence.J Mol.ApplGenet.1,561-573), the selection marker gene is hygromycin phosphotransferase gene (hpt). Its T-DNA region such as figure 1 shown.

[0043] Using the plasmid containing the SiLEA14 gene as a template, use the PCR method to obtain a SiLEA gene fragment with Hind III and Spe I restriction sites at both ends, connect it to the pMD19T vector, and perform doubl...

Embodiment 3

[0044] Example 3 Construction and Agrobacterium Transformation of Expression Vectors for SiLEA14 Gene Expression in Millet

[0045] The expression cassette is composed of the maize Ubiquitin promoter and the 3' transcription termination region from the nopaline synthase (nos) gene (Depicker A, Stachel S., Dhaese P, Zam bryski P, Goodman HM (1982). Nopalinesynthase: transcript mapping and DNA sequence.J Mol.Appl Genet.1,561-573), the selectable marker gene is hygromycin phosphotransferase gene (hpt). Its T-DNA region such as figure 2 shown.

[0046] Using the plasmid containing the SiLEA14 gene with a flag-tag tag as a template, the SiLEA gene fragment with BamH I and Kpn I restriction sites at both ends was obtained by PCR method, connected to the pMD19T vector, and sequenced correctly with BamH I and Kpn I Kpn I was subjected to double enzyme digestion, and a fragment of about 600bp was recovered. The expression vector pCOU (containing maize Ubiquitin promoter) was double...

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Abstract

The present invention clones for the first time the SiLEA14 gene (Seq ID No.1) related to plant adversity stress, and transforming the gene into plants can improve plant stress resistance. The invention provides a new method for increasing stress resistance of plants by using SiLEA14 gene, which can be used for breeding new stress-resistant varieties of plants and has important significance for agricultural production.

Description

technical field [0001] The invention relates to the field of plant genetic engineering, in particular to the millet SiLEA14 gene and its application. Background technique [0002] Environmental stresses such as drought and high salinity can cause serious damage to crops, resulting in a significant drop in yield. In order to adapt to unfavorable environmental conditions, plants have formed a series of defense mechanisms in the long-term evolution process. Among them, dependent on Ca 2+ In the signaling pathway of LEA, the activation of Late Embryogenesis Abundant (LEA) gene can protect plants from damage (Xiong L, Schumaker KS, Zhu J (2002) Cell signaling during cold, drought and salt stress. Plant Cell14 ,S165-S183). [0003] The LEA gene was first discovered in cotton seeds (Dure L and Galau GA (1981) Developmental biochemistry of cotton seed embryogenesis and germinationXIII.Regulation of biosynthesis of principal storage proteins.PlantPhysiol.68,187-194), expressed in ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/29C12N15/84C12N1/21A01H5/00C12Q1/68C12N15/11
Inventor 于静娟王梅珍朱登云李萍
Owner CHINA AGRI UNIV
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