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Protein PbrTTS1 with pollen tube growth promoting function of Dangshan pears and coding gene and application of protein

A technology of Dangshansu pear and coding gene, which can be applied in the application, genetic engineering, plant genetic improvement and other directions, can solve the problem of no pollen tube growth and the like

Active Publication Date: 2018-11-16
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

And there is no report about the genes controlling pollen tube growth in pears in the prior art

Method used

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  • Protein PbrTTS1 with pollen tube growth promoting function of Dangshan pears and coding gene and application of protein
  • Protein PbrTTS1 with pollen tube growth promoting function of Dangshan pears and coding gene and application of protein
  • Protein PbrTTS1 with pollen tube growth promoting function of Dangshan pears and coding gene and application of protein

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preparation example Construction

[0063] In the present invention, the preparation method of the accelerator preferably comprises the following steps:

[0064] It is 10% sucrose that will contain mass concentration, the boric acid of mass concentration 0.01%, the calcium nitrate of mass concentration 0.03%, the solution dissolution above-mentioned protein of the 2-morpholineethanesulfonic acid (MES) of 0.03mmol / L, adjust pH with Tris .

[0065] The invention provides the application of the protein, the coding gene PbrTTS1, the primer or the pollen tube growth promoter in pollination or pollen tube growth.

[0066] In the present invention, the method of said pollination preferably comprises the following steps:

[0067] a. remove the stamens from the Dangshansu pears on the day before the big bud stage, and obtain the stamenless Dangshansu pear flowers;

[0068] b. Spray the protein on the stigma of the Dangshansu pear flower, dip the pollen of the non-Dangshansu pear variety on the stigma of the Dangshansu ...

Embodiment 1

[0073] Tissue localization of PbrTTS1 gene

[0074] RNA was extracted from the stem, leaf, pulp, pollen, and style of 'Dangshan Suli', and the first-strand cDNA obtained by reverse transcription was used to detect the expression site of PbrTTS1.

[0075] For RNA extraction, a Total Plant RNA Extraction Kit (purchased from FOREGENE, operated according to the operating instructions provided by the kit) was used. The first-strand cDNA was synthesized with a TransScript reverse transcription kit (purchased from Beijing Quanshijin Biotechnology Co., Ltd., and operated according to the instructions provided by the kit). Amplified gene primer pair: PbrTTS1-F1: 5'-TGTCTTCGTTCACCCACCAG-3' (SEQ ID No.7); PbrTTS1-R1: 5'-CGCTACAAAGCTCCTTGGGA-3' (SEQ ID No.8), and PbrTubulin as an internal reference gene , the primer pair is PbrTubulin-F:5'-TCAGTCGCCGCCGGCCTTTTG-3'(SEQ ID No.9); PbrTubulin-R:5'-TGGGCTTTGCTCCTCTTAC-3'(SEQ ID No.10). 20μL PCR reaction system includes 100ng cDNA, 2× Hieff ...

Embodiment 2

[0078] Identification of polymorphisms in PbrTTS1 gene

[0079] RNA was extracted from the styles of 'Dangshansuli', 'Fengshui', 'New Century', 'Cuiguan', 'Huanghua' and 'Xishui', and the first-strand cDNA obtained by reverse transcription was used for PbrTTS1 gene clone.

[0080] RNA extraction and reverse transcription were carried out according to Example 1. The amplification gene primer pair is SEQ ID No.3 and SEQ ID No.4. The high-fidelity enzyme for cloning genes uses Q5 High-Fidelity DNA Polymerase (purchased from NEB Company). The 50 μL PCR reaction system includes 2 μL cDNA, 10 μL 5×Q5 Reaction Buffer, 1 μL 10 mmol / L dNTPs, 0.5 μL Q5 High-Fidelity DNA Polymerase, 10 μmol / L Add 2.5 μL of each primer and make up to 50 μL with water. The PCR reaction was completed on a Veriti amplification instrument according to the following program: 98°C, pre-denaturation for 3 s, denaturation at 98°C for 10 s, annealing at 66°C for 30 s, extension at 72°C for 20 s, 35 thermal cycles...

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Abstract

The invention provides a protein PbrTTS1 with a pollen tube growth promoting function of Dangshan pears and a coding gene and application of the protein and belongs to the technical field of genetic engineering of plants. A recombinant protein is used for culturing pollen so as to treat pollen tubes, a result proves that the protein can promote the growth of the pollen tubes, and thus, a regulation and control mechanism that a non-S factor in the pears participates in an SI process is extended. Meanwhile, the recombinant protein is adopted to research the mechanism that the non-S factor participates in the SI process, the labor cost can be reduced to a great extent, the efficiency of pollination is increased, and the protein has very important theoretical and practical significance in agricultural production.

Description

technical field [0001] The invention belongs to the technical field of plant genetic engineering, and specifically relates to a Dangshansu pear protein with the function of promoting pollen tube growth, a coding gene PbrTTS1 and applications thereof. Background technique [0002] The phenomenon of self-incompatibility (SI) is a common way in the plant kingdom to control inbreeding (Iwano and Takayama 2012). SI mainly includes three types: sporophytic self-incompatibility, poppyceae self-incompatibility and gametophytic self-incompatibility based on S-RNase. Among them, gametocyte self-incompatibility based on S-RNase mainly includes Rosaceae, Solanaceae, and Plantainaceae species (Clarke and Newbigin 1993; Huang et al.2009). [0003] Pear belongs to the species of Rosaceae, which mainly exhibits the phenomenon of SI based on S-RNase. In the SI response of pear, the S-locus genotypes of female and male gametes played a decisive role. When the genotype carried by the male g...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/415C12N15/29C12N15/11C12N15/10A01N47/44A01P21/00A01H1/02
CPCA01N47/44C12N15/10C07K14/415C12Q2531/113C12Q1/6895C12P19/34
Inventor 张绍铃焦慧君吴巨友许林林黄小三谢智华刘倩常耀军王鹏
Owner NANJING AGRICULTURAL UNIVERSITY
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