A method for cloning and transient expression of genes related to persimmon deastringency

一种脱涩、基因的技术，应用在基因工程、植物基因改良、植物学设备和方法等方向，能够解决调控机制未见报道等问题

Active Publication Date: 2014-10-29
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Yamada et al. (2002) believed that ADH and PDC are the key enzymes of acetaldehyde synthesis in persimmon fruit, but so far, the regulatory mechanism of ADH and PDC in the study of persimmon fruit astringency has not been reported

Method used

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  • A method for cloning and transient expression of genes related to persimmon deastringency
  • A method for cloning and transient expression of genes related to persimmon deastringency
  • A method for cloning and transient expression of genes related to persimmon deastringency

Examples

Experimental program
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Embodiment 1

[0024] Example 1: Real-time quantitative fluorescent PCR analysis of the expression patterns of ADH and PDC family members in persimmons in different treatments;

[0025] (1) Experimental method

[0026] 1. Extraction of total RNA from persimmon fruit

[0027] At the designed sampling points, control, ethylene and carbon dioxide treated fruit pulp were frozen in liquid nitrogen and stored at -80°C. When extracting RNA, weigh 2g of the frozen sample and add liquid nitrogen to fully grind it, then add it to two centrifuge tubes with 4ml β-mercaptoethanol / CTAB (80ul / 4ml) extraction buffer, heat at 65°C, and vortex to mix the cells Completely rupture, heat again at 65°C for 1-2min; then add 4ml of chloroform:isoamyl alcohol (24:1) extract to the centrifuge tube, vortex quickly to mix evenly; centrifuge at 10,000rpm at 15°C for 10min, absorb the supernatant to a new Extract the centrifuge tube with chloroform:isoamyl alcohol (24:1) again, absorb about 3ml of the supernatant, comb...

Embodiment 2

[0033] Example 2: Construction of transient expression system in persimmon leaves.

[0034] (1) Experimental method Obtaining the full-length sequences of ADH and PDC gene family members in persimmon fruit:

[0035] Firstly, 5'RACE primers were designed according to the obtained 3' end sequence, and the 5'RACE (rapid amplification of cDNA ends) cloning technology system was used to use the 5'RACE cDNA as a template, and finally the 5' RACE gene family members of ADH and PDC were obtained. terminal sequence. Then use the 5' end sequence as a template to design upstream primers and downstream primers spanning the initiator and terminator respectively, using tissue cDNA as a template, PCR reaction system: template cDNA: 1ul, DNTP mix (2.5mM): 1.6ul, Upstream primer (10mM): 0.8ul, downstream primer (10mM): 0.8ul, Pyrobest DNA Polymerase: 0.1ul, 10×pyrobest Buffer: 2.0ul, DEPC water: 13.7ul. Amplify by PCR: pre-denaturation at 94°C for 5 minutes, 35 cycles (95°C for 30 s, 55°C fo...

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Abstract

The invention provides a cloning and transient expression method of persimmon deastringency related genes. The method comprises: first acquiring gene 3'-terminal sequences, then employing 3' RACE (rapid amplication of cDNA ends) technology to acquire 3'-terminal sequences of ADH and PDC gene family members in persimmon fruits; adopting Q-PCR (quantitative polymerase chain reaction) technology to analyze the genetic expression of ADH and PDC gene family members in a persimmon fruit deastringency treatment; and conducting separation to obtain full-length sequences of ADH and PDC gene family members, then establishing a transient expression system of persimmon leaves. The method of the invention clones the persimmon deastringency related gene family members, analyze the expression patterns of the family members in the persimmon deastringency treatment, and finally verifies functions of the target genes through transient expression of the persimmon leaves. The invention clarifies the regulation mechanism for postharvest persimmon deastringency from the molecular mechanism aspective, and can be used for further functional verification (transgenosis, gene interaction and the like) study of the postharvest persimmon deastringency mechanism.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and relates to the cloning and expression of genes related to persimmon deastringency and the establishment of a system for transient expression of persimmon leaf genes. Background technique [0002] Persimmon is native to China. It is delicious and good for human health. It is one of the most popular fruits in East Asia (China, Japan, Korea, etc.). The unique feature of persimmon is that its fruit can accumulate tannins (PAs) during development. ), tannin is a polymer substance, and soluble tannin is the main substance that presents the astringency of persimmon fruit. [0003] Persimmons can be divided into four categories according to their deastringency type and whether the seeds affect the astringency and color of the fruit: complete sweet persimmon (PCNA); complete astringent persimmon (PCA); partial sweet persimmon (PVNA); partial astringent persimmon (PVA). ). Sweet persimmon fr...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/53C12N15/10C12Q1/68C12N15/82
Inventor 殷学仁闵婷陈昆松孙崇德
Owner ZHEJIANG UNIV
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