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Chimonanthus praecox CpFPA gene as well as encoded protein and application thereof

A wax plum and protein technology, which can be used in applications, genetic engineering, plant genetic improvement, etc., can solve problems such as unclear molecular mechanisms

Active Publication Date: 2021-02-09
SOUTHWEST UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in Wintersweet, there are few studies on the genes involved in the regulation of flowering through autonomous pathways, and the specific molecular mechanism in Wintersweet is still unclear.

Method used

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  • Chimonanthus praecox CpFPA gene as well as encoded protein and application thereof
  • Chimonanthus praecox CpFPA gene as well as encoded protein and application thereof
  • Chimonanthus praecox CpFPA gene as well as encoded protein and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1 Cloning and Molecular Characterization of Wintersweet CpFPA Gene

[0034] Carry out sequence analysis on the CpFPA gene obtained from the transcriptome to design specific primers, use the first strand of C. cDNA as a template to amplify the CpFPA gene. The PCR reaction system and reaction conditions are as follows:

[0035]

[0036] Using CpFPA specific primers to carry out PCR amplification, the specific product with a size of about 3100bp is amplified from the cDNA of Wintersweet, as figure 1 shown. The PCR products were respectively ligated with pMD19-T and transformed into Escherichia coli competent cells, and the monoclonal liquid with the correct band identified by PCR was sent to Huada Company for sequencing. The sequence is shown in SEQ ID No.1.

[0037] Through EditSeq, Vector NTI 9.0 software to analyze the cDNA sequence characteristics of Chimera CpFPA gene, it was found that the full-length cDNA sequence of Wintersweet CpFPA gene was 3626 nucle...

Embodiment 2

[0040] Example 2 Analysis of the expression characteristics of Wintersweet CpFPA gene

[0041] The tissues and complete floral organs of different periods of adult Chimonanthus praecox ‘grandiflorus’ that were routinely maintained and managed on the campus of Southwest University were selected for total RNA extraction and reverse transcription into cDNA.

[0042] When carrying out real-time fluorescent quantitative (qPCR) analysis on the CpFPA gene of Chimera japonica, the Actin gene and Tublin gene of Chimera japonica were used as double internal reference genes, and the internal reference gene and target gene-specific primers were designed using the software PrimerPremier5.0. The specific primer sequences See Table 1.

[0043] Table 1 Wintersweet real-time fluorescent quantitative PCR primers

[0044]

[0045]

[0046] Note: F stands for upstream primer, R stands for downstream primer.

[0047] Using the first strand of cDNA obtained by reverse transcription of the R...

Embodiment 3

[0056] Example 3 Wintersweet CpFPA Gene Expression Vector Construction and Plant Genetic Transformation

[0057] In order to ensure that the target gene is inserted into the plant expression vector without being affected by enzyme digestion, the restriction site and distribution characteristics of the largest ORF frame sequence of the CpFPA gene were analyzed, and combined with the multiple cloning of the plant expression vector pCAMBIA1300 According to site characteristics, the most suitable enzymes KpnI and XbaI for double digestion were screened out. In order to amplify the coding regions of the CpFPA gene carrying KpnI and XbaI restriction sites for subsequent experiments, the restriction sites and their corresponding protective bases were designed to be added upstream and downstream of the original specific primer sequences. The primer names and sequences are as follows:

[0058] p-CpFPA-F( The underlined part is the protected base, and the boxed part is the KpnI restr...

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Abstract

The invention relates to the field of plant molecular biology, in particular to a chimonanthus praecox CpFPA gene, and an encoded protein and application thereof. According to the chimonanthus praecoxCpFPA obtained by cloning, the maximum open read frame (ORF, open read frame) of a cDNA sequence is 3099bp, and 1032 amino acids are encoded. Arabidopsis thaliana inflorescence is dip-dyed with pCAMBIA1300-CpFPA, it is found that compared with wild type arabidopsis thaliana (Col-0), transgenic arabidopsis thaliana has the advantages that the expression quantity of AtFLC and AtSVP is remarkably reduced, the expression quantity of AtAP1 and AtLFY is increased, and the scape time, bud appearing time, first flowering time, and first capsule forming time of transgenic arabidopsis thaliana strainsare all earlier than those of Col-0, rosettes are reduced, stem leaves are increased, and early blossoming phenotypes are presented. The result shows that the CpFPA gene can inhibit the expression ofAtFLC so as to promote the early blossoming of plants.

Description

technical field [0001] The invention belongs to the field of plant molecular biology, and specifically relates to a wintersweet CpFPA gene, its coded protein and application. Background technique [0002] Wintersweet (Chimonanthus praecox) is a deciduous shrub with long flowering period and aromatic smell. It is not only a unique traditional precious flower in my country, but also endowed with profound historical culture since ancient times, with high ornamental value and humanistic value. Wintersweet is native to central my country and is now widely distributed, including Sichuan, Chongqing, Hunan, Zhejiang and other places. [0003] With the rapid development of molecular biology, the research on the molecular aspects of wintersweet has gradually become a hot spot. Existing studies include the analysis of genetic diversity and variety classification, stress resistance, and analysis of the functions of flowering-related genes through molecular markers, in order to maximize...

Claims

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Application Information

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IPC IPC(8): C07K14/415C12N15/29C12N15/82A01H5/00A01H6/20
CPCC07K14/415C12N15/827
Inventor 李志能乌春雨眭顺照李名扬
Owner SOUTHWEST UNIVERSITY
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