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PDF1 gene for controlling plant pollen fertility, and preparation method and application thereof

A technology of fertility and inhibitory factors, which is applied in the field of plant genetic engineering and biology, can solve the problems of low expression level, no other genes found, poor gene conservation, etc.

Active Publication Date: 2011-02-16
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

But so far no other genes involved in the PCD pathway have been identified in other plant species
This may be due to the low expression level of gene transcripts involved in PCD, or the poor conservation of these genes involved in PCD (Roger I. Pennell and Chris Lamb. Programmed Cell death in plants. The plant cell, Vol.9, 1157-1168.1997)

Method used

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  • PDF1 gene for controlling plant pollen fertility, and preparation method and application thereof
  • PDF1 gene for controlling plant pollen fertility, and preparation method and application thereof
  • PDF1 gene for controlling plant pollen fertility, and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0083] Cloning of PDF1 homologous genes and BnPDF1 expression pattern analysis:

[0084] 1. Cloning of PDF1 homologous gene:

[0085] Carry out the extraction of total RNA according to the requirement of Trirol extraction kit (mentioned above), specific method is as follows: get respectively 0.05-0.1g of Brassica oleracea, Brassica napus, Chinese cabbage-type rape leaf sample, grind to powder in liquid nitrogen, RNA was extracted according to the requirements of the Trirol extraction kit. The extracted total RNA was dissolved in 60uL of RNase-free double distilled water. DNase I (described above) removes possible residual DNA. A protein detector (DU 650 BECKMAN, 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

[0108] Example 2.: microRNA-PDF1 vector construction, Arabidopsis transformation and PCR detection:

[0109] 1. Vector construction of microRNA-PDF1

[0110]The vector was constructed according to the method listed in "2010 Project of Arabidopsis" (http: / / wmd3.weigelworld.org / cgi-bin / webapp.cgi?page=Home; project=stdwmd). The selected target sequence is: AATUTUTATATUUTGTUGAG, and the corresponding Oligo DNA is: TTAGAGACTATAGGACAGCTC. Carrier structure such as image 3 shown.

[0111] Then microRNA-PDF1 was transferred into Agrobacterium tumefaciens EHA105 (purchased from Dalian Bao Biological Products Co., Ltd., hereinafter the same) using the freeze-thaw method (described above), and coated with 50 μg / mL ampicillin and rifampicin ( 50 μg / mL) of LB solid (recipe is as follows: Weigh 10 grams of tryptone, 5 grams of yeast extract and 10 grams of sodium chloride, and 8 grams of agar are dissolved in distilled water in turn, and the volume is set at 1000 ml. Packed in 500 In ...

Embodiment 3

[0126] Example 3: Phenotype observation of transgenic Arabidopsis:

[0127] During the flowering period of Arabidopsis, select the flowers that have leaked at the top of each branch but have not yet opened, and use a dissecting needle to poke the flower buds. Anthers and pollen were observed under a magnifying glass (3 times) and a stereomicroscope (OLYMPUS SZ61TRC). Anthers were isolated with a dissecting needle, and the anthers soaked in Alexander's staining solution were heated overnight in a 40°C water bath (Alexander, M.P. Differential staining of aborted and non-aborted pollen. As mentioned above), the microspores were observed under a microscope (OLYMPUS IX71) by the tablet method. The degree of abortion of microspores was determined by observing and counting the ratio of normal microspores (stained orange) and aborted microspores (stained green). Arabidopsis anther frozen section and hematoxylin staining methods refer to the Arabidopsis floral organ frozen section te...

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Abstract

The invention discloses a protodermal factor 1 (PDF1) gene for controlling plant pollen fertility, and a preparation method and application thereof. A separated protein is obtained from cabbage, cabbage type rape and turnip type rape respectively, wherein sequences of the proteins are nucleotide sequences shown by SEQ ID No. 1, SEQ ID No. 2 and SEQ ID No. 3 respectively. The nucleotide sequences are coding sequences (CDS) which are cloned by taking cDNA as a template. A separated polypeptide is obtained from the cabbage, the cabbage type rape and the turnip type rape respectively, wherein the sequences of the separated polypeptides are amino acid sequences shown by SEQ ID No. 4, SEQ ID No. 5 and SEQ ID No. 6 respectively. The invention discloses the application of the PDF1 gene for controlling the plant pollen fertility, and relates to isolation and cloning, expression analysis and application of the PDF1 homologous gene for controlling microspore development and anther fertility. ThePDF1 is a programmed cell death (PCD) suppression factor; and the gene has obvious expression advantage in rape anther. By weakening the expression of the PDF1 gene through gene engineering technology, the microspore can be aborted, the fertility of the pollen can be controlled, a male sterile material and a restorer material can be created, and the gene can be used for development research of androgamete and improvement on quality of farm crops.

Description

technical field [0001] The invention relates to the fields of plant genetic engineering and biotechnology. It specifically relates to a PDF1 gene for controlling the fertility of plant pollen, and also relates to a preparation method for the PDF1 gene for controlling the fertility of plant pollen, and also relates to an application of the PDF1 gene for controlling the fertility of plant pollen. Background technique [0002] PDF1 (protodermal factor 1) gene has a full length of 1.421kp and exists on the second chromosome of Arabidopsis thaliana. About 57 of the 306 amino acids encoded by the PDF1 gene are prolines, which is a proline-rich protein. Usually this protein can form a special quaternary structure and interact with other proteins to play a regulatory role. It has been reported that the expression of some cotton proline-rich proteins (proline-rich proteins, PRPs) has developmental stage and tissue specificity, and the expression of these genes is also affected by pa...

Claims

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Application Information

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IPC IPC(8): C12N15/29C07K14/415A01H5/00
Inventor 刘胜毅王琢玉董彩华李振波黄军艳刘越英
Owner INST OF OIL CROPS RES CHINESE ACAD OF AGRI SCI
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