Rape BnPABP 5 gene and application of promoter thereof

A promoter and gene technology, applied in the field of plant genetic engineering and biology, can solve the problems of abnormal anther development and male sterility in plants

Inactive Publication Date: 2011-07-06
INST OF OIL CROPS RES CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Through transgenic experiments, it is proved that inhibiting the expression of thi

Method used

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  • Rape BnPABP 5 gene and application of promoter thereof
  • Rape BnPABP 5 gene and application of promoter thereof
  • Rape BnPABP 5 gene and application of promoter thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0078] Cloning and expression pattern analysis of a highly expressed gene BnPABP5 in rapeseed anthers:

[0079] 1. Cloning of a high-efficiency expression gene BnPABP5 in rapeseed anthers

[0080] Carry out the extraction of RNA according to the requirement of Trirol extraction kit (mentioned above), specific method is as follows: get the root, stem, leaf, flower, silique sample of 0.05-0.1g respectively, grind to powder in liquid nitrogen, according to Trirol extraction kit is required for RNA extraction. The extracted total RNA was dissolved in 60uL of RNase-free double distilled water. DNase I removes possible residual DNA. A protein detector (DU 650BECKMAN, USA) was used to detect the light absorption values ​​of RNA at 260 nm and 280 nm, respectively, and the purity and concentration of RNA were identified by 1% (mass volume ratio) agarose gel electrophoresis. Use the RNA obtained above as a template to carry out reverse transcription according to the following scheme:...

Embodiment 2

[0091] A kind of preparation of rapeseed BnPABP5 promoter:

[0092] 1. Cloning of rape BnPABP5 gene promoter:

[0093] Utilize SDS cracking method to extract rapeseed DNA (J. Sambrook. D.W. Russell works, Huang Peitang et al. Translate Molecular Cloning Test Guide (Third Edition) Science Press), operate according to genome walking kit (purchased in Clontech company) According to the procedure, two genome walks are carried out, and two rounds of PCR amplification are carried out for each walk, and the promoter (3688bp) of the rapeseed BnPABP5 gene is cloned. The primers used for the two walks are listed below.

[0094] Table 1 Primers used for genome walking cloning of rapeseed BnPABP5 promoter

[0095]

[0096] The specific steps are as follows:

[0097]According to the instructions of the Genome Walking Kit (purchased from Clontech, Universal.GenomeWalker), about 2.5 μg of genomic DNA was digested in a 100 μl system with the restriction endonucleases DraI, EcoRV, PvuII ...

Embodiment 3

[0100] Arabidopsis transformation and PCR detection of pBI121-BnPABP5:

[0101] Arabidopsis thaliana was transformed according to the transformation method in the above literature (Zhang X R. et al. Agrobacterium-mediated transformation of Arabidopsis thaliana using the floral dip method. Nature, 2006, 1:1-6). According to the unique kanamycin resistance of the transgenic plants, they were grown on 1 / 2MS (mentioned above) medium containing 50 mg / L kanamycin, and the green shoots obtained were preliminarily considered as positive shoots. After the green seedlings grew two true leaves, they were transplanted into vermiculite. After the plants appeared inflorescences, a true leaf was taken to extract genomic DNA by SDS method for positive PCR identification. The primer sequence sequence is 5′-AAAAGACTTACCGAGTTGAACC-3′ and 5′-CGCTGTCGTTGGGGCAATTC-3′; the PCR reaction system is as follows: Genomic DNA template 1μL (about 50ng), 10×Taq enzyme reaction buffer 2ul, 25mM MgCL 2 1.2ul,...

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Abstract

The invention discloses a rape BnPABP 5 gene and the application of a promoter thereof. The sequence of a segregation gene is nucleotide sequence indicated by SEQ ID No. 1, SEQ ID No.2. The promoter comprises a recombinant vector. The invention also discloses the application of the rape BnPABP 5 gene the promoter thereof in controlling pollen fertility and anther. The gene and the promoter thereof are predominantly expressed in the rape anther. The invention further relates to the isolation, cloning, analysis of expression and application of gene BnPABP5 and the promoter thereof for controlling microspore development and the anther fertility. The BnPABP 5 gene has the function of controlling the anther development of rape as well as the pollen fertility. The expression of the BnPABP 5 gene can be weakened through the technique of gene engineering so that the pollen fertility can be controlled and male sterile line materials as well as restorer line materials can be created, therefore, the rape BnPABP 5 gene can be applied to the development and research and the quality improvement of rape andro gamete. The promoter of the BnPABP 5 gene is only predominantly expressed in the anther, and the promoter with tissue preference has higher potential of edible safety on transgenic oil crops, thus having certain development and application value.

Description

technical field [0001] The invention relates to the fields of plant genetic engineering and biotechnology. It specifically relates to a gene related to pollen development, a promoter controlling the high-efficiency expression of the gene in pollen or anther, and a preparation method of the gene and the promoter. The present invention also relates to a vector containing the gene and its promoter or its homologous nucleotide sequence and relates to the application of the gene or its promoter in rapeseed genetic engineering. Background technique [0002] Most eukaryotic mature mRNA molecules have a poly(A) tail, which is related to the stability of mRNA and the regulation of translation initiation. Poly(A) binding protein (poly(A)binding protein, PABP) widely exists in different types of eukaryotic cells, and has a strong affinity with 25bp or longer poly(A) (Sachs A B, Davis R W and Kornberg R.D.A single domain of yeastpoly(A)-binding protein is necessary and sufficient for ...

Claims

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Application Information

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IPC IPC(8): C12N15/29C12N15/11C12N15/82A01H5/02
Inventor 刘胜毅石磊董彩华黄军艳刘越英
Owner INST OF OIL CROPS RES CHINESE ACAD OF AGRI SCI
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