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Gene participating in forming of peach fruit combined state linalool and application thereof

A combination state, linalool technology, applied in application, genetic engineering, plant genetic improvement, etc., can solve the problem of lack of transgenic system

Active Publication Date: 2018-07-06
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Genome sequencing results show that peach contains at least 160 members of the UGT gene family, coupled with the lack of transgenic systems, how to identify and clone UGTs involved in the synthesis of bound linalool is a major technical challenge

Method used

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  • Gene participating in forming of peach fruit combined state linalool and application thereof
  • Gene participating in forming of peach fruit combined state linalool and application thereof
  • Gene participating in forming of peach fruit combined state linalool and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Embodiment 1: PpUGT85A2 gene cloning

[0021] (1) Experimental method

[0022] Taking the amino acid sequence of AdGT4 with UDP-glucosyltransferase function in kiwifruit as the reference sequence, the blastp algorithm was used to search for the homologous sequence of peach in the peach genome database Peach Genome V2.0, select the sequence with the highest matching degree, and design primer pairs For SEQ:NO.2 and SEQ:NO.3, using peach cDNA as a template, PCR amplification was performed to obtain the PpUGT85A2 sequence SEQ:NO.1, which was then verified by sequencing. The PCR reaction system was 50 μl, and the components were: 0.5 μl Taq enzyme (Roche), 5 μl buffer (10×), 4 μl dNTP (2.5 mM), 2 μl each of upstream and downstream primers (10 μM, Invitrogen), 4 μl cDNA, 32.5 μl H 2 O. The RT-qPCR reaction program was 95°C for 5 min; 95°C for 30 s, 58°C for 30 s, and 72°C for 2 min, 37 cycles; finally, 72°C for 10 min and storage at 4°C.

[0023] (2) Experimental results ...

Embodiment 2

[0025] Example 2: There is a positive correlation between the expression of PpUGT85A2 in peach fruit and the content of bound linalool linalyl-β-D-glucoside

[0026] (1) Experimental method

[0027] 1. Peach fruit material

[0028] Peach fruits were harvested at the hard core stage (71DAB), rapid fruit expansion stage (94DAB) and ripe stage (108DAB). Ripe fruits were stored at 20°C for 3 days until fully ripe. At the same time, the harvested ripe peach fruits were treated with ethylene (Ethylene) to accelerate fruit ripening, 1-methylcyclopropene (1-MCP) to delay ripening, and air treatment as control (Control). The pulp tissue was frozen in liquid nitrogen and stored in a -80°C refrigerator until use. Three biological replicates were set up, each with 5 fruits.

[0029] 2. RNA Extraction and cDNA Synthesis

[0030] The total RNA of peach fruit was extracted by CTAB method, and after genomic DNA contamination was removed by TURBO DNase Kit (Ambion) kit, 1.0 μg of RNA was ...

Embodiment 3

[0038] Example 3: Escherichia coli heterologous expression of PpUGT85A2 recombinant protein catalyzes the synthesis of bound linalool linalyl-β-D-glucoside

[0039] (1) Experimental method

[0040] 1. Recombinant vector construction and E. coli transformation

[0041] Combined with the primer pair SEQ:NO.4 and SEQ:NO.5, PpUGT85A2 was amplified by PCR technology, and the PCR system and reaction procedure were the same as in Example 1. The PCR product and the target vector (pET6xHN-N, Clonetch, Takara) were digested with restriction endonucleases SalI and HindIII and ligated for sequencing verification. The plasmid was transformed into Escherichia coli competent BL21(DE3)pLysS(Promega) by heat shock method , Pick positive colonies for PCR detection and verification.

[0042] 2. Induced expression

[0043] Pick a single colony, add it to 20 mL of LB containing ampicillin antibiotics, and culture overnight at 37°C. After overnight culture, the bacterial solution was transferre...

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Abstract

The invention provides a gene PpUGT85A2 participating in forming of peach fruit combined state linalool. A full-length sequence of PpUGT85A2 is obtained through PCR (polymerase chain reaction) amplification, and the situation that PpUGT85A2 is clustered in a UGT85 family is obtained through analyzing an evolutionary tree. An in-vitro functional verification conducted in escherichia coli shows thatPpUGT85A2 can convert linalool to be in a combined state mode without the volatility under the condition that UDP-glucose serves as a sugar donor. PpUGT85A2 is overexpressed in a peach fruit and tobacco, the content of linalyl-beta-D-glucoside in combined state linalool is outstandingly increased, storage of fragrant matter is increased, and thus the gene has important application value on improving the fragrance quality of processed food by adopting a hydrolase to release the combined state linalool in the processing process of agricultural products after being picked.

Description

technical field [0001] The invention belongs to the fields of plant molecular biotechnology and genetic engineering, and relates to a gene involved in the formation of peach fruit bound linalool and its application. Background technique [0002] Volatile compounds usually exist in plants in two forms: free state and glycoside-bound state. Free forms of volatile compounds are freely released from the plant. Combined aroma substances are not volatile and have no aroma themselves. Studies have shown that combined aroma substances are abundant in fruits and are an important reserve form of aroma substances. These bound aroma substances can be released into free aroma substances through hydrolysis during ripening or post-harvest processing, which can significantly increase the aroma of fruits or foods. Linalool has a floral scent and can exist in a conjugated form. Therefore, identifying the genes that form substances such as bound linalool has important potential application...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/54C12N15/11C12N15/82A01H5/00A01H6/82A01H6/74
CPCC12N9/1051C12N15/8243C12Y204/01017
Inventor 张波吴伯萍陈昆松
Owner ZHEJIANG UNIV
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