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Protein related to pollen germination and/or pollen tube growth, coding gene and use thereof

A technology for pollen germination and encoding genes, which is used in applications, genetic engineering, plant genetic improvement, etc.

Inactive Publication Date: 2009-08-12
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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  • Protein related to pollen germination and/or pollen tube growth, coding gene and use thereof
  • Protein related to pollen germination and/or pollen tube growth, coding gene and use thereof
  • Protein related to pollen germination and/or pollen tube growth, coding gene and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

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

[0037] 1. Obtaining the middle sequence of the gene PwTUA1

[0038]In the sunny morning when the inflorescences of Qinggan (Zhang LY, Lin JX, et al. Plant Science, 2007, 172: 1210-1217.) are open, use sterilized tweezers to remove the yellow pollen lumps and put them on white clean paper Then put it into a sterilized dry glass bottle, put it in the room to dry in the shade, then pack it into a dry glass bottle, seal it, and store it in a -20°C refrigerator for later use. Total RNA was extracted from Pseudomonas pollen, and cDNA was obtained by reverse transcription. Using the cDNA as a template, degenerate primers were designed according to the highly conserved regions of the tubulin-encoding genes of Arabidopsis, rice, cotton and poplar published in GenBank 5'-GATGCHTTCWACACHTTYTTYAG-3' and 5'-AGGGCRGCVAGRTCCTCACG-3', were PCR amplified. In the desi...

Embodiment 2

[0053] Example 2, analysis of tissue expression specificity of PwTUA1 gene by semi-quantitative RT-PCR

[0054] Extract the RNA from the pollen, needle leaf, bark and root tissue of the green stem, reverse transcribe and synthesize cDNA; The primers were used for PCR amplification to detect the expression of PwTUA1 gene in different tissues of C. At the same time, the EF1-α gene was used as an internal reference, and the 5' primer for amplifying the EF1-α gene was: 5'-AACTGGAGAAGGAACCCAAG-3', and the 3' primer was: 5'-AACGACCCAATGGAGGATAC-3'.

[0055] PCR reaction conditions: pre-denaturation at 94°C for 5 minutes; then 10 sec at 94°C, 10 sec at 56°C, 20 sec at 72°C, a total of 28 cycles; extension at 72°C for 5 min.

[0056] The expression of PwTUA1 gene in each tissue is as follows: figure 1 shown. The results showed that the PwTUA1 gene was expressed only in pollen, but not in other tissues.

Embodiment 3

[0057] Example 3, Cultivation of transgenic blue stem pollen and Arabidopsis with high pollen germination rate and pollen tube growth rate

[0058] 1. Construction of expression vector

[0059] The plasmid pBI121 (purchased from Clontech Company) and the plasmid pLat52-7 containing the Lat52 promoter (David Twell, Rod Wing, Judy Yamaguchi et al. Isolation and expression of an anther-specific gene from tomato, Mol Gen Genet, 1989, 217: 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.) were carried out with restriction endonuclease HindIII and BamHI Double enzyme digestion, and then connect the digestion products to obtain the recombinant vector PBI121-1; take 2 μl of the PwTUA1 gene obtained in the above-mentioned Example 1 and connect it to the pMD18-T vector, and the operation steps are carried out according to the instructions of the produ...

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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, its coding gene and application. Background technique [0002] Pollen germination and pollen tube growth are important steps in the sexual reproduction of seed plants. Seed plants must rely on pollen tubes to transfer sperm and egg cells to fuse and complete fertilization. The metabolic activity in the pollen tube is very active, with a high synthesis rate of protein, lipid and carbohydrate, all of which are involved in the expansion of the top of the pollen tube and the synthesis of new cell walls. Pollen tube is a typical apical growth cell, and its growth is directional, so pollen tube provides a special model system for experimental research. The research results on pollen and the cytoskeleton in the pollen tube are mostly obtained from angiosperms as experimental materials. Due to the differences in the evolution of gymnosperms and angiosperms, the proces...

Claims

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Application Information

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Patent Type & Authority Applications(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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