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Gene transient expression method of citrus peel

A technology of transient expression and citrus peel, applied in the field of bioengineering, can solve the problems of long cycle, complicated operation, and difficulty in obtaining positive transgenic plants, and achieve the effect of reasonable design and convenient and simple operation

Inactive Publication Date: 2016-09-28
ZHEJIANG UNIV
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Problems solved by technology

[0005] The purpose of the present invention is to provide a gene transient expression method of citrus peel, to overcome the shortcomings of existing citrus genetic transformation methods such as complex operation, long cycle, and difficulty in obtaining positive transgenic plants, etc., and to solve the homology of genes related to citrus peel Functional verification, providing new ideas and methods for functional verification of gene homology in perennial fruit trees

Method used

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  • Gene transient expression method of citrus peel
  • Gene transient expression method of citrus peel
  • Gene transient expression method of citrus peel

Examples

Experimental program
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Effect test

Embodiment 1

[0015] Embodiment 1: Citrus peel CitERF13 gene is transiently expressed in sweet orange peel

[0016] 1. Extraction of RNA and synthesis of cDNA: the extraction of total RNA from different tissues and organs of citrus (stem, leaf, flower, fruit) was carried out by CTAB method, and TURBO DNase (Ambion, USA) was used to remove genomic DNA contamination, and then 1.0 μg RNA was reverse-transcribed into cDNA with iScript cDNA Synthesis Kit (Bio-Rad, USA) for future use, and specific operations were performed according to the instructions.

[0017] 2. Construction of expression vectors: Based on the Sweet Orange and Clementine Citrus Genome Database (http: / / www.citrusgenomedb.org), BLAST (TBLASTN and BLASTP) analysis to obtain CitERF13 (SEQ: NO.1) related to chlorophyll degradation. The gene was amplified by using PCR technology and primer pair SEQ: NO.2 and SEQ: NO.3, and further verified by sequencing means. The open reading frame of the gene was amplified according to the conf...

Embodiment 2

[0020] Example 2: Transient expression of Citrus peel CitERF13 gene in Ponkan peel

[0021] Whole steps are the same as in Example 1, and the injected fruit is the ponkan peel.

[0022] Transient expression experiment was carried out by taking Ponkan fruit 160 days after the full flowering stage. After 5 days of injection, the pericarp phenotype and chlorophyll content are as attached figure 2 , compared with the control, the degradation of chlorophyll in the pericarp injected with CitERF13 was significantly enhanced. The samples of pericarp tissue in the Agrobacterium-infiltrated area were collected, and it was found that the chlorophyll content of the pericarp injected with CitERF13 was significantly lower than that of the negative control

[0023] The present invention uses the citrus genome database to isolate and obtain CitERF13 related to chlorophyll degradation, load it on the pGreen II 0029 62-SK expression vector, and introduce it into Agrobacterium GV3101::pSoup, ...

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Abstract

The invention provides a gene transient expression method of citrus peel. The gene transient expression method comprises the following steps: carrying out RNA extraction and cDNA synthesis on different tissue organs of citrus; loading exogenous target genes to a pGreen II 002962-SK carrier, and electrotransferring the obtained carrier to agrobacterium GV3101::pSoup, and analyzing the transient expression of the citrus peel by adopting a formula of penetrating fluid and a unique injection method and combining with strict contrast. The method is reasonable in design, and convenient and easy to operate, and can be used for rapidly carrying out homologous functional verification on genes of the perennial citrus tree, and therefore, a novel thought and a novel method for provided for the homologous functional verification of the genes of the perennial fruit tree. The method is also suitable for the study on the transient expression of other fruits.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and relates to a gene transient expression method of citrus peel, which can be used for homologous verification of gene functions of perennial fruit trees. Background technique [0002] Citrus belongs to Rutaceae (Rutaceae) citrus subfamily (Aurantioideae), divided into Poncirus Raf (Poncirus Raf), Golden Citrus (Fortnoella Swing) and Citrus L. (Citrus L.), is the world's largest fruit tree species, its planting area and The output ranks first, and it is also one of the original fruits in my country. Citrus pulp has a unique flavor and has anti-cancer and anti-tumor nutritional functions, and citrus peel is rich in aromatic substances and natural pigments, which is a good material for studying volatile substances and pigment metabolism and its regulation related genes. [0003] Gene function verification can be divided into two situations, one is to enhance its expression and obtain expres...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/84
CPCC12N15/8261
Inventor 殷学仁陈昆松谢秀兰刘晓芬
Owner ZHEJIANG UNIV
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