Cabbage pollen wall growth related gene PGBc2 and separation method thereof

A technology of pollen wall and gene, applied in the field of genetic engineering

Inactive Publication Date: 2008-09-03
ZHEJIANG UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

No genes or mutants that act on pollen wall development have been isolated in Chinese cabbage

Method used

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  • Cabbage pollen wall growth related gene PGBc2 and separation method thereof
  • Cabbage pollen wall growth related gene PGBc2 and separation method thereof
  • Cabbage pollen wall growth related gene PGBc2 and separation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1 Isolation and cloning of PGBc2 gene

[0037] (1) Isolation of Bc-A8T18 Fragment Using A8 / T18 primers, cDNA-AFLP analysis of gene expression differences between sterile lines and fertile lines of Chinese cabbage ajhGMS'Bcajh97-01A / B' was detected. The gene band Bc-A8T18 specifically expressed in flower buds was used to cut off the differential fragments on the polyacrylamide gel with a scalpel blade, and dissolved in 100 μL TE (10 mmol·L -1 Tris, pH 7.5, 1mmol L -1 EDTA, pH 8.0), and used as a template for PCR amplification under the same conditions as pre-amplification in cDNA-AFLP. PCR products were subjected to 1.5% agarose gel electrophoresis and purified by a gel recovery kit. The recovered product was connected to pGEM-T Easy Vector (Promega) and transformed into E.coli DH 5α competent cells. The recombinant plasmid was screened by blue and white spots, sequenced, and finally the nucleotide sequence of the band was verified by RT-PCR.

[0038] (2) The...

Embodiment 2

[0040] Example 2 Using antisense RNA technology to verify the function of PGBc2

[0041] (1) Construction of antisense expression vector According to the full-length sequence of PGBc2 cDNA, primers were designed between the 85th base and the 546th base in the PGBc2 sequence, and the 462bp sequence was amplified as an antisense gene fragment, see SEQ ID No :3. And according to the multiple cloning enzyme cutting site of the plant binary vector pBI121, design two PCR amplification primers containing BamH I and Xba I restriction endonuclease sites and three protective bases respectively, and the upstream primer is 5'- ACA GGATCC GAGCTTTTACCGTTTTTA-3', the downstream primer is 5'-GCC TCTAGA TGGCATCTATTGAAGATA-3' (the underlined part is the enzyme cutting site). The cDNA of flower buds of 'Bcajh97-01B' was used as a template for PCR amplification. The product was cloned into pGEM-T Easy vector, and the positive cloned plasmid was named pTPGBc2A.

[0042] (2) Construction of ...

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Abstract

The invention discloses a growth of Chinese cabbage pollen wall related gene PGBc2 and separation thereof. The upgrowth of Chinese cabbage pollen wall related gene includes nucleotide sequence indicated in SEQ ID No. 2. Separation clone and functional verification of the gene is helpful to illustrate molecular mechanism in growth of pollen and male sterility of plants, and has practical meaning for artificially controlling fertility of plant, cultivating superior varieties.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a Chinese cabbage pollen wall development-related gene PGBc2 and an isolation method thereof. Background technique [0002] Pollen development is closely related to plant male sterility, and it is an ideal model for studying processes such as cell growth, division, cell differentiation, and intercellular information transmission. Therefore, the research on pollen development has always been a hot spot in the field of life science research. The process of pollen development is complex and involves the expression regulation of many genes. Studies have found that a major feature of pollen gene expression is the expression of a large number of genes related to cell wall synthesis and regulation. The most characteristic part of the pollen grain is the pollen wall (Blackmore and Barnes, 1990; Owen and Makaroff, 1995). It is composed of two layers, the outer wall of the pollen and ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/29C12N15/10
Inventor 黄鹂曹家树叶意群张豫超张爱红
Owner ZHEJIANG UNIV
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