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Key ethylene response factor for regulating chlorophyll degradation of citrus peels

A technology of citrus peels and response factors, which can be used in applications, genetic engineering, plant genetic improvement, etc., and can solve problems such as unclear biological mechanisms

Inactive Publication Date: 2016-10-26
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the biological mechanism by which ERFs family members regulate chlorophyll degradation during citrus peel degreening remains unclear.

Method used

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  • Key ethylene response factor for regulating chlorophyll degradation of citrus peels
  • Key ethylene response factor for regulating chlorophyll degradation of citrus peels
  • Key ethylene response factor for regulating chlorophyll degradation of citrus peels

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1: Gene Cloning

[0026] CitAP2 / ERF gene family members were obtained based on the Citrus Sweet Orange and Clementine Genome Database (http: / / www.citrusgenomedb.org). On the one hand, directly download the genes that may belong to AP2 / ERF according to the annotations, on the other hand, perform BLAST (TBLASTN and BLASTP) analysis on the reported Arabidopsis AP2 / ERF genes, and use the sequences obtained in these two ways to use AP2 / ERF The conserved domains were screened one by one, and then the CAP3 sequence assembly program (http: / / pbil.univ-lyon1.fr / cap3.php) was used to remove repetitive sequences to obtain CitERF6 (SEQ: NO.1) and CitERF8 (SEQ: NO. .2).

[0027] Combining the primer pair SEQ: NO.3 and SEQ: NO.4, and SEQ: NO.5 and SEQ: NO.6, use PCR technology to amplify the two genes obtained respectively, wherein the 30 μl PCR system is: 10×Buffer 3 μl , dNTP 2.4 μl, upstream / downstream primers 0.6 μl each, enzyme 0.15 μl, DEPC-H 2 O 21.3 μl, 50 mM MgSO ...

Embodiment 2

[0028] Example 2: Gene expression analysis

[0029] (1) Experimental method

[0030] 1. Fruit material collection

[0031]The navel orange fruits 150 days after the full flowering period arrived at the laboratory on the same day after harvest, and the fruits with uniform size and relatively consistent maturity were selected to carry out the experiment. The fruits were randomly divided into two groups (100 in each group), and each group was placed in 3 airtight containers on average, and one group was passed through 40 μL·L -1 Ethylene gas for 12 hours, while the other group was ventilated with air for 12 hours, and then transferred to a shelf and placed in an environment of 20°C. Samples were taken at 0, 4, 8, 12 and 48 hours respectively. Nine fruits were taken each time and divided into three biological replicates.

[0032] 2. Determination of the color index (CCI) of citrus peel: it is determined by the color difference meter MiniScan XE Plus (HunterLab, USA), and the c...

Embodiment 3

[0040] Example 3: Verification of gene function

[0041] (1) Experimental method

[0042] 1. Construction of recombinant vector

[0043] Primers (SEQ: NO.3 and SEQ: NO.4) were designed according to the verified full-length sequence of CitERF6 (SEQ: NO.1) to amplify its open reading frame. Using the cDNA of citrus peel as a template, slightly modified the high-fidelity enzyme system of Roche Company, and prepared a PCR system with a final volume of 30 μl: 10×Buffer 3 μl, dNTP 2.4 μl, upstream / downstream primers 0.6 μl, enzyme 0.15 μl, DEPC-H 2 O2 1.3 μl, 50 mM MgSO 4 1.2 μl, cDNA 0.75 μl. The reaction conditions were: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 10 sec, annealing at 58°C for 5 sec, extension at 72°C for 2.5 min, and 35 thermal cycles; extension at 72°C for 10 min, and storage at 4°C. After the PCR product was recovered, it was connected to the pGEM-T easy vector, and the recombinant plasmid was introduced into E. coli DH5α competent cells, ...

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Abstract

The invention provides a key ethylene response factor CitERF6 (SEQ: NO. 1) for regulating the chlorophyll degradation of citrus peels. The key ethylene response factor comprises a conservative AP2 / ERF structural domain and a Pat4(RKRK) nuclear localization signal. During the ethylene treatment of harvested citrus fruits, the expression of CitERF6 is enhanced under external ethylene induction. When the CitERF6 instantly overexpresses in the citrus peels, the chlorophyll degradation of the citrus peels can be promoted and accelerated. The key ethylene response factor has the advantages that by the PCR, real-time and quantitative PCR and citrus fruit instant expression technologies, the citrus ethylene response factor CitERF6 is cloned, and identification of the CitERF6 shows that the CitERF6 can participate in the chlorophyll degradation; the CitERF6 is applicable to genetic engineering breeding of plant color modification and especially to the regulation of the chlorophyll degradation of the citrus peels, and a theoretical basis is provided for the further researches of the peel coloring technology and the acting mechanism of the CitERF6.

Description

technical field [0001] The invention belongs to the fields of plant molecular biotechnology and genetic engineering, and relates to a key ethylene response factor (CitERF6) for regulating the degreening of citrus peels, and its application in genetic engineering breeding of plant color modification and in regulating the degreening of citrus peels. 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 functions, while the peel is rich in aromatic substances and natural pigments (chlorophyll and carotenoids). Citrus fruits are often harvested during the green ripening period. In order to promote the uniform coloring of t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/29C12N15/84A01H5/08
CPCC07K14/415C12N15/8249
Inventor 陈昆松谢秀兰刘晓芬殷学仁
Owner ZHEJIANG UNIV
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