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A protein related to pollen germination and/or pollen tube growth, its coding gene and application

A technology for pollen germination and coding genes, which is applied in application, genetic engineering, plant genetic improvement, etc.

Inactive Publication Date: 2012-02-08
BEIJING FORESTRY UNIVERSITY +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Tubulin plays an important role in the germination and polar growth of plant pollen. At present, the research on the role of microtubules in pollen germination and pollen tube growth is limited to the protein level, and there is no research on the level of gene transcription regulation.

Method used

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  • A protein related to pollen germination and/or pollen tube growth, its coding gene and application
  • A protein related to pollen germination and/or pollen tube growth, its coding gene and application
  • A protein related to pollen germination and/or pollen tube growth, its coding gene and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1. Obtaining of protein related to pollen germination and / or pollen tube growth and its coding gene PwTUA1

[0037] 1. Obtain the intermediate sequence of gene PwTUA1

[0038] Yu Qingzhu (Zhang LY, Lin JX, et al. Plant Science, 2007, 172: 1210-1217.) Sunny morning when the inflorescences are open, use sterilized tweezers to remove the yellow pollen block and place it on white clean paper Put it in a sterilized dry glass bottle, dry it indoors, and then divide it into dry glass bottles, seal them, and store them in a refrigerator at -20℃ for later use. Extract the total RNA of Qingzhu pollen, reverse transcription to obtain cDNA. Using the cDNA as a template, design degenerate primers based on the highly conserved regions of Arabidopsis, rice, cotton and poplar tubulin coding genes published in GenBank 5'-GATGCHTTCWACACHTTYTTYAG-3' and 5'-AGGGCRGCVAGRTCCTCACG-3' for PCR amplification. In the designed primers, H represents A, T or C, W represents A or T, Y represents...

Embodiment 2

[0057] Example 2. Analysis of tissue expression specificity of PwTUA1 gene by semi-quantitative RT-PCR

[0058] The RNA from the pollen, needles, bark and root tissues of Qingzhu was extracted, and cDNA was synthesized by reverse transcription. Then, the cDNA of the above-mentioned tissues was used as the template, and 5'-TGCATGATCTCCAATTCGAC-3' and 5'-GCACTGTTCTCAACATGAAG-3' For PCR amplification with primers, the expression of PwTUA1 gene in different tissues of Qingzhu was detected. At the same time, using EF1-α gene as an internal reference, the 5'primer for amplifying EF1-α gene is: 5'-AACTGGAGAAGGAACCCAAG-3', and the 3'primer is:

[0059] 5’-AACGACCCAATGGAGGATAC-3’.

[0060] PCR reaction conditions: pre-denaturation at 94°C for 5 minutes; then at 94°C for 10sec, 56°C for 10sec, and 72°C for 20sec, 28 cycles in total; then extension at 72°C for 5 minutes.

[0061] The expression of PwTUA1 gene in various tissues such as figure 1 Shown. The results showed that only the pollen ha...

Embodiment 3

[0062] Example 3 Cultivation of transgenic Qingzhu pollen and Arabidopsis with high pollen germination rate and pollen tube growth rate

[0063] 1. Construction of expression vector

[0064] Plasmid PBI121 (purchased from Clontech) and plasmid pLat 52-7 (David Twell, Rod Wing, Judy Yamaguchi et al. Isolation and expresstion of an anther-specific gene from tomato, Mol Gen Genet, 1989, 217) containing the Lat52 promoter : 240~245; David Twell, Theodore MKlein, Michael E Fromm et al. Transient expression of chimeric genes delivered intopollen by microprojectile bombardment. Plant Physiol, 1989, 91: 1270~1274.) Restriction enzymes HindIII and BamHI were used respectively Carry out double enzyme digestion, and then ligate the digested products to obtain the recombinant vector PBI121-1; take 2μl of the PwTUA1 gene obtained in Example 1 above and ligate it with the pMD18-T vector. The operation steps follow the instructions of Promega's product pMD18-T Vector system Proceed; the obtained...

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Abstract

The invention discloses a protein related to pollen grain germination and / or pollen tube growth, the encoding genes and the application thereof. The protein is the following protein shown in (1) or (2): (1) the protein being composed of amino acid sequences shown in sequence two of a sequence table; (2) the protein comprises the amino acid in the sequence two of the sequence table substituted and / or lacked and / or added and is related to the stress tolerance of plant. The protein and the encoding gene thereof related to the pollen grain germination and / or pollen tube growth can be used for the genetic transformation research of the plant, increases the pollen germination rate of plant and / or the growth speed of the pollen tube and the reproductive efficiency of the plant, shortens the breeding process, has important theoretical and practical actual meaning for adjusting and controlling the reproductive development and gene engineering breeding of woody plants with slow growth development and accelerating the genetic improvement of the woody plants.

Description

Technical field [0001] The invention relates to a protein related to pollen germination and / or pollen tube growth and its coding gene and application. Background technique [0002] Pollen germination and pollen tube growth are important links in the sexual reproduction of seed plants. Seed plants must rely on pollen tubes to transfer sperm and egg cells to fuse to complete fertilization. The metabolic activity in the pollen tube is very active, with a high synthesis rate of protein, lipids and carbohydrates. These substances are involved in the top expansion of the pollen tube and the synthesis of new cell walls. The pollen tube is a typical apical growth cell, and its growth is directional. Therefore, the pollen tube provides a special model system for experimental research. The research results on pollen and the cytoskeleton in the pollen tube are mostly observed using angiosperms as experimental materials. Due to the evolutionary difference between gymnosperms and angiosperm...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/415C12N15/29C12N15/63C12N5/10C12N1/00C12N15/11C12N15/82A01H5/00C12N1/15C12N1/19C12N1/21
Inventor 张凌云于彦丽李彦泽郑成超
Owner BEIJING FORESTRY UNIVERSITY
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