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Pear flowering regulation transcription factor PbrSPL15 and application thereof

A transcription factor and protein technology, applied in the field of plant genetic engineering, can solve problems such as lower fruit setting rate, unstable temperature, and prone to freezing damage, and achieve the effects of accelerating the breeding process, earlier plant flowering, and reducing agricultural production costs

Active Publication Date: 2020-02-25
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Pear trees generally bloom in spring, but the temperature in spring is extremely unstable, and frost damage is prone to occur, resulting in a decrease in fruit setting rate

Method used

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  • Pear flowering regulation transcription factor PbrSPL15 and application thereof
  • Pear flowering regulation transcription factor PbrSPL15 and application thereof
  • Pear flowering regulation transcription factor PbrSPL15 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0066] Cloning of Full-length cDNA of PbrSPL15 Gene in Pear

[0067] A flowering transcription factor PbrSPL15 was screened by screening the full-length pear cNDA library. According to the recombination characteristics of the Gateway system and the sequence of the PbrSPL15 gene, primers were designed with Primer Premier 5.0, and the whole pear was amplified by PCR using the cDNA of the phloem of branches of 'Dangshansuli' as a template. long. The detailed steps are as follows:

[0068] The research material Dangshansu pear is planted in the National Pear Engineering Center of Nanjing Agricultural University, and its seedling age is 3 to 5 years. The annual branches of Dangshansu pear with strong growth potential were selected, and 500 mg samples were randomly weighed, and immediately frozen with liquid nitrogen. The CTAB method was used to extract total RNA. Before the experiment, prepare RNA-free blue tips, yellow tips, white tips and 1.5ml centrifuge tubes; Quick freeze w...

Embodiment 2

[0071] qRT-PCR Analysis of PbrSPL15 Gene Regulating Flowering Transcription Factor in Dangshansu Pear Branches of Different Ages

[0072] In order to analyze the response pattern of PbrSPL15 gene to age in Dangshansu pear, the expression pattern of PbrSPL15 gene was analyzed using Real-time PCR technology. According to the sequence of the coding region of the PbrSPL15 gene, according to the general principles of primer design, primer primer 5.0 software was used to design the upstream and downstream PCR primers for amplifying the entire coding region of the gene. The kit was used to extract RNA, and the synthesis of the first strand of cDNA was carried out according to the operation manual of Thermo Scientific RevertAid First Strand cDNA Synthesis Kit. The 20 μL reaction system includes: 10 μL SYBR Green, 5 μL sterilized ultrapure water, 1 μL cDNA, 2 μL forward primer, F2: 5'-GTCGTAGGCGTTTGTCAGGA-3' (SEQ ID No.5), 2 μL reverse primer, R2: 5 '-AGCCTTTCCACAGCATGAGAC-3' (SEQ ID ...

Embodiment 3

[0077] Subcellular Localization of PbrSPL15 Protein Regulating Flowering Transcription Factor in Pear

[0078] According to the nucleotide sequence of the PbrSPL15 gene and the maps of the TOPO vector and the pMDC83-GFP vector, EcoRI and XhoI restriction sites were added before and after the gene sequence. The sequence of the restriction site is as follows:

[0079] EcoRI: GAATTC

[0080] XhoI: CTCGAG

[0081] Using the cDNA of the phloem of Dangshansu pear branches as a template, amplified with primers added restriction sites, the PCR program used was: pre-denaturation at 98°C for 2 minutes; denaturation at 98°C for 10 seconds, annealing at 58°C for 15 seconds, extension at 72°C for 1 minute, 35 Cycle; 72°C extension for 10 min. The primer sequences of the restriction sites are as follows:

[0082] F1: 5'-CACC GAATTC ATGGAGGGGATGAGTAAATTG-3' (SEQ ID No. 3)

[0083] R1: 5'- CTCGAG TTTGATTTGGAAGTGCTTGT-3' (SEQ ID No. 4)

[0084] The stop codon TAG was removed from the...

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Abstract

The invention discloses a pear flowering regulation transcription factor PbrSPL15. A nucleotide sequence of the pear flowering regulation transcription factor PbrSPL15 is as shown in SEQ ID No. 1, orthe pear flowering regulation transcription factor PbrSPL15 has more than 90% of homology with the SEQ ID No.1, and is the DNA molecule for regulating a plant flowering related protein is encoded. Thepear flowering regulation transcription factor PbrSPL15 provided by the invention can regulate plants to bloom in advance, shorten the juvenile phase and provide new gene resources, and is beneficialto accelerating the breeding process, reducing the agricultural production cost, and improving the economic benefit.

Description

technical field [0001] The invention belongs to the technical field of plant genetic engineering, and relates to the construction of pear flowering regulating transcription factor PbrSPL15 and its recombinant vector and the application in regulating plant flowering ability. Background technique [0002] Plants are completely divided into two stages: vegetative growth and reproductive growth. Higher plants need a certain period of vegetative growth after germination before they can enter reproductive growth. Flowering is a sign of plant transition from vegetative growth to reproductive growth. For most plants, proper flowering time is crucial for their survival and normal reproduction, and is directly related to crop yield and economic benefits. As an important trait in plant life history, flowering time plays a central role in plant production and evolution (Luo Rui and Guo Jianjun, 2010). [0003] Due to differences in their own characteristics, different plants experienc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/415C12N15/82A01H5/00A01H6/46
CPCC07K14/415C12N15/8205C12N15/827
Inventor 吴巨友曹鹏王鹏张绍铃刘哲吴潇孔佳君黄智齐开杰谢智华
Owner NANJING AGRICULTURAL UNIVERSITY
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