Capsella bursa-pastoris CBF pathway key gene and application in cultivation of cold resistant plant thereof

A gene, shepherd's purse technology, applied in the fields of molecular biology and genetic engineering, can solve problems such as few successful examples and no cold-resistant plants have been found, and achieve the effect of improving cold resistance and great academic and economic value.

Inactive Publication Date: 2010-12-22
FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Although there have been many attempts to improve the cold tolerance of plants by using transgenic technology, so far, there are not many successful examp

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Cloning of the key gene CbCBF and CbCOR gene of embodiment 1 shepherd's purse CBF pathway

[0040] 1. Cultivation of shepherd's purse seedlings

[0041] The shepherd's purse seeds come from Shanghai Seed Company. After the shepherd's purse seeds are sterilized, they are sown in the 0 The culture tank of the culture medium was placed at 25° C. for 4 weeks, and the photoperiod was 16 hours of light and 8 hours of darkness.

[0042] 2. Isolation of DNA

[0043] Take shepherd's purse leaves, add liquid nitrogen, grind into powder, and use CTAB method to extract DNA. The quality of DNA was identified by agarose gel electrophoresis, and then the content of DNA was determined on a spectrophotometer.

[0044] 3. Cloning of genes

[0045] According to the known amino acid coding regions of the CBF and COR genes of shepherd's purse (GenBank Accession No.: AY391121 and GenBank Accession No.: AY437888), two pairs of primers were designed, wherein, a pair of (5'-ATGTCTTTCTCAGGAG...

Embodiment 2

[0046] Cloning of the promoter sequence of the key gene CbCOR gene of embodiment 2 shepherd's purse CBF pathway

[0047] The promoter sequence of CbCOR gene of shepherd's purse was amplified by chromosome walking technique. Experimental operation according to UniversalGenomeWalker TM Kit (CLONTECH) manual. After the chromosome walking library of shepherd's purse was built, two primers (5'-TACTCCGCTGTGGAAAGAAGAACCC-3' and 5'-GTCATCGAGGATGTTGCCGTCACCT-3', denoted as SEQ ID NO.6) were designed according to the sequence of SEQ ID NO.1. The two linker primers are combined to carry out two rounds of PCR reactions, and the two rounds of PCR amplification products are detected by electrophoresis at the same time. The specific bands in the second round were selected for cloning and sequencing. Sequencing results showed that the promoter sequence of the cloned CbCOR gene was 1180bp in full length. See SEQ ID NO.2.

[0048] The promoter sequence of the obtained shepherd's purse was...

Embodiment 3

[0049] The analysis of the key gene CbCBF and CbCOR gene of embodiment 3 shepherd's purse CBF pathway

[0050] The length of the shepherd's purse CbCBF sequence cloned in the present invention is 660bp, and the detailed sequence is shown in SEQ ID NO.3, wherein the open reading frame is located at 1-660 nucleotides. Compared with the cDNA sequence of shepherd's purse CbCBF, the similarity can reach 100%, indicating that there is no intron in the nucleotide sequence of shepherd's purse CbCBF. The length of the shepherd's purse CbCOR sequence cloned in the present invention is 837bp, and the detailed sequence is shown in SEQ ID NO.1, wherein the open reading frame is located at 1-191 and 612-837 nucleotides. By comparing with the shepherd's purse CbCOR cDNA sequence, the sequence similarity of the coding region can reach 100%, but there is a 420bp intron in the CbCOR sequence.

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PUM

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Abstract

The invention belongs to the technical field of molecular biology and genetic engineering, in particular to a capsella bursa-pastoris CBF path key gene and application in cultivation of a cold resistant plant thereof. In the invention, capsella bursa-pastoris is used as an experimental material, a nucleotide sequence and a promoter of a COR gene (a key gene in the CBF pathway of cloning the capsella bursa-pastoris by a PCR technique) and the nucleotide sequence of the CBF gene, which are represented by SEQ ID No. 1, SEQ ID No. 2 and SEQ ID No.3 respectively, are used to construct a plant express vector, and the cold resistant transgenic plant is obtained by agrobacterium-mediated genetic transformation. By fully exploring potential gene resources of the capsella bursa-pastori and using the gene resources for improving crop variety, the invention has great economic value.

Description

technical field [0001] The invention belongs to the technical fields of molecular biology and genetic engineering. Specifically, the present invention relates to a key gene CBF (CRT / DRE binding factor) gene and COR ( co ld- r egulated) genes and promoters. After these genes are transferred into plants, the CBF protein expressed in the plants can activate the promoter of the COR gene, thereby increasing the expression of the COR gene, and finally regulating the cold resistance of the plant. Background technique [0002] Plants have sufficient adaptability and resistance to environmental changes and adverse environments. This stress resistance is not only controlled by the genetic type of phylogenetic evolution, but also restricted by physiological and ecological factors in individual development. As one of the important environmental factors, temperature limits the distribution, growth and yield of plants, and plays a decisive role in plant growth and development (Viswanath...

Claims

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Application Information

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IPC IPC(8): C12N15/29C12N15/11C12N15/82
Inventor 林娟周明琦吴丽华
Owner FUDAN UNIV
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