Rape BnPAB5 gene and application thereof

A technology of gene and rapeseed pollen, applied in the field of plant genetic engineering and biology, can solve the problem of less research on higher plants

Inactive Publication Date: 2013-04-17
INST OF OIL CROPS RES CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Studies on PAB genes are mainly concentrated in yeast and animals, and their primary structure and gene structure are conserved, while studies on higher plants are less

Method used

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  • Rape BnPAB5 gene and application thereof
  • Rape BnPAB5 gene and application thereof
  • Rape BnPAB5 gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0103] Cloning and expression pattern analysis of a highly expressed gene BnPAB5 in rapeseed anthers:

[0104] 1. Cloning of a high-efficiency expression gene BnPAB5 in rapeseed anthers

[0105] The rapeseed used in the present invention is Brassica napus L. Zhongshuangjiu (provided by Associate Researcher Wang Xinfa of Oil Crops Research Institute, the same below). Zhongshuang No. 9 was sown in Daejeon and managed in the field normally. Rapeseed young leaf samples were ground into powder in liquid nitrogen, and RNA was extracted according to the requirements of the Trirol extraction kit (described above). The extracted total RNA was dissolved in RNase-free double distilled water. DNase I (purchased from Promega, the same below) removed possible residual DNA. A protein detector (DU650BECKMAN, USA) was used to detect the light absorbance of RNA at 260 nm and 280 nm, and the purity and concentration of RNA were identified by 1% (mass volume ratio) agarose gel electrophoresis....

Embodiment 2

[0117] Functional analysis of the BnPAB5 gene:

[0118] 1. Construction of BnPAB5 gene RNA interference plant expression vector RNAi-AtPAB5:

[0119] In order to study the function of the pollen highly expressed gene BnPAB5 in the present invention, RNA interference technology was used. Since rapeseed BnPAB5 and Arabidopsis AtPAB5 have similar expression patterns, gene structures (described above), and more than 76% protein homology, it is speculated that they have the same function. Therefore, the function of rape PAB5 was obtained by studying the function of Arabidopsis AtPAB5 gene. On the basis of the above sequence analysis, the CDS sequence of the AtPAB5 gene was selected with the highest homology to the CDS sequence of the BnPAB5 gene. A 400bp sequence located in the second exon of Arabidopsis is the target fragment of RNA interference. From this, primers PAB5-Ri-1-1 and PAB5-Ri-1-2 (described above) were designed. The 5' ends of the primer sequences introduced XbalI...

Embodiment 3

[0135] Complementary function verification of BnPAB5 gene:

[0136] 1. Highly efficient and specific expression of promoter P in anthers BnPABP3 Construction of plant expression vector OX-BnPAB5 driven by BnPAB5 gene overexpression and transformation of Agrobacterium tumefaciens strain EHA105.

[0137] In order to further confirm that the rapeseed BnPAB5 gene and the Arabidopsis AtPAB5 gene have the same function, we constructed the highly efficient and specific expression promoter P BnPABP3 (Patent No.: ZL2011110122101.2, Invention Name: Brassica napus BnPABP3 promoter preparation method and its application, as described above) BnPAB5 gene overexpression driven by plant expression vector OX-BnPAB5, transformed into Arabidopsis thaliana. The obtained OX-BnPAB5 transgenic offspring were used as the male parent, and the RNA interference RNAi-AtPAB5 male sterile transgenic offspring was used as the female parent to perform hybridization to detect whether the anther fertility of ...

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Abstract

The invention discloses a rape BnPAB5 gene and application of the rape BnPAB5 gene. A separated gene has the sequence as shown in SEQ ID No.1 and SE1 ID No.2. The PAB5 in combination with the poly(A) tail plays a special role in the transcript processes, such as starting of polyadenylation, transporting and translation, thus the expression processes of other genes can be controlled. The BnPAB5 shows preferential expression in rape anther and microspore. The expression of Arabidopsis AtPAB5 is interfered to result in abortion of the microspore. The transgenic arabidopsis of overexpression BnPAB5 is utilized as a male parent and pollinated to the transgenic arabidopsis of the interference AtPAB5, and the fertility of plant anther of filial generation and pollen can be recovered. As the complementary test shown, the rape BnPAB5 and the arabidopsis AtPAB5 have the same functions, such as the functions of controlling the development of the rape anther and arabidopsis anther and the fertility of the pollen; the expression level of the PAB5 can be controlled by genetic engineering technology, thus the fertility of the pollen can be controlled, and male sterility line materials and restoring line materials can be created; and the rape BnPAB5 gene can be applied to the study on male gametes development and the improvement of quality of crop.

Description

technical field [0001] The invention relates to the fields of plant genetic engineering and biotechnology. It specifically relates to a PAB5 gene for controlling the pollen fertility of rapeseed and Arabidopsis thaliana, a preparation method for the PAB5 gene for controlling the pollen fertility of plants, and an application of the PAB5 gene for controlling the pollen fertility of plants. The present invention also relates to a carrier containing the gene or its homologous nucleotide sequence and an application of the gene in genetic engineering of rapeseed. 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, PAB) 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 yeast poly(A)-binding pro...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/29C07K14/415
Inventor 刘胜毅董彩华黄军艳石磊童超波刘越英程晓辉
Owner INST OF OIL CROPS RES CHINESE ACAD OF AGRI SCI
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