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Critical ethylene response factor (ERF) for regulating and controlling orange peel chlorophyll removal and application of critical ERF

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

Inactive Publication Date: 2015-07-15
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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  • Critical ethylene response factor (ERF) for regulating and controlling orange peel chlorophyll removal and application of critical ERF
  • Critical ethylene response factor (ERF) for regulating and controlling orange peel chlorophyll removal and application of critical ERF
  • Critical ethylene response factor (ERF) for regulating and controlling orange peel chlorophyll removal and application of critical ERF

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Example 1 : Gene Cloning

[0022] 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 annotation, on the other hand, perform BLAST (TBLASTN and BLASTP) analysis on the reported Arabidopsis AP2 / ERF gene, and use the sequences obtained by these two methods 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 CitERF13 (SEQ: NO. 1) and CitERF8 (SEQ: NO . 2).

[0023] Combining the primer pair of SEQ: NO. 3 and SEQ: NO. 4, and SEQ: NO. 5 and SEQ: NO. 6, using 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...

Embodiment 2

[0024] Example 2 : Gene expression analysis

[0025] (1) Experimental method

[0026] 1. Fruit material collection

[0027] The fruits of Newhall navel orange in the developing stage were harvested at 90, 105, 120, 135, 150, 165 and 180 days after the full flowering stage. Nine fruits were collected each time and divided into three biological replicates.

[0028] The ponkan fruits 160 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 for experiments. The fruits were randomly divided into two groups (100 in each group), and each group was placed in 3 airtight containers on average, and a group of 40 μL·L -1 Ethylene gas was used for 12 h, while the other group was ventilated with air for 12 h, and then placed in a shelf at 20 °C. The two groups of fruits were sampled at 0, 4, 8, 12 and 48 hours of treatment, respectively. Nine fruits were taken ea...

Embodiment 3

[0038] Example 3 : Gene function verification

[0039] (1) Experimental method

[0040] 1. Recombinant vector construction

[0041] Primers (SEQ: NO. 3 and SEQ: NO. 4) were designed according to the verified full-length sequence of CitERF13 (SEQ: NO. 1) to amplify its open reading frame. Using the cDNA of citrus peel as a template, slightly modify the high-fidelity enzyme system of Invitrogen Company, prepare 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 O 21.3 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 ce...

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Abstract

The invention provides a critical ethylene response factor (ERF) CitERF13 for regulating and controlling orange peel chlorophyll removal. The gene contains a conservative AP2 / ERF structural domain and a Pat4 (RKRK) core positioning signal. During the development of orange fruits, the expression peak of the CitERF13 is degraded in advance of peel chlorophyll, and is enhanced by exogenous ethylene. When the CitERF13 is excessively expressed instantaneously in orange peels, the degradation of chlorophyll can be promoted, and the peel chlorophyll removal is accelerated. A technology of PCR (polymerase chain reaction), real-time quantitative PCR and orange fruit instantaneous expression is utilized to clone and identify the critical ERF CitERF13 which can be used for regulating and controlling the chlorophyll degradation, and can be applied to plant color modified genetic engineering breeding especially to regulation and control of orange peel chlorophyll removal.

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 (CitERF13, SEQ: NO. 1) regulating citrus peel degreening, and its application in genetic engineering breeding for plant color modification. Background technique [0002] Citrus belongs to Rutaceae (Rutaceae) citrus subfamily (Aurantioideae), divided into Poncirus Raf (Poncirus Raf), Golden Citrus (Fortnoella Swing), Citrus L. (Citrus L.), is the world's largest fruit tree species, its planting area It is also one of the original fruits in my country. Citrus pulp has a unique flavor and has anti-cancer and anti-tumor properties, while the peel is rich in aromatic substances and natural pigments (chlorophyll and carotenoids). Chlorophyll not only affects the photosynthesis of fruit, but also plays a major role in fruit coloration. Among many citrus varieties, some gradually degrade the chlorophyll content in the per...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/29C12N15/10C12N15/82A01H5/08
Inventor 陈昆松谢秀兰殷学仁刘晓芬孙崇德徐昌杰
Owner ZHEJIANG UNIV
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