哈密瓜果实转录因子CmCBF1的克隆及表达分析

doi:10.6048/j.issn.1001-4330.2017.10.001

摘要/Abstract

摘要： 【目的】研究CBF在哈密瓜果实采后抗冷过程中的调控功能，对转录因子CmCBF₁进行克隆与表达分析，为进一步研究哈密瓜果实低温贮藏过程中的冷害发生机制提供理论依据。【方法】以新密3号采后离体果实为试材，根据已报道的甜瓜CBF1预测序列(XM_008440940)，与基因组甜瓜组(https://melonomics.net)进行BLAST比对，确定基因编码区起始和终止位置，针对CBF₁编码区设计特异性引物，通过对果实cDNA进行扩增，获得CBF1全长序列，利用实时荧光定量PCR(qRT-PCR)分析其在不同温度条件下的表达特性。【结果】克隆获得哈密瓜果实转录因子CBF1，命名为CmCBF₁，该基因序列为639 bp(GenBank登录号为KT737742)，编码212个氨基酸，蛋白质分子量为23.89 kDa，等电点为5.23;同源性分析表明，哈密瓜果实转录因子CmCBF₁与草莓FaCBF1亲缘关系较近，达到85%。qRT-PCR分析结果显示，哈密瓜果实转录因子CmCBF₁在1、3及5℃低温诱导条件下特异性表达，在20℃室温条件下几乎不表达。【结论】转录因子CmCBF₁的表达与哈密瓜果实采后冷害发生负相关(r=-0.663)。

关键词: 哈密瓜; CmCBF₁; 克隆; 表达

Abstract: 【Objective】 In order to make clear the regulatory function of CBF gene in the cold tolerance of Hami melon fruits, the transcription factor named CmCBF₁ was cloned and expression analysis was carried out.【Method】'Xinmi No.3' Hami melon postharvest fruit was selected as materials. The primers used to amplify the full-length coding sequences were designed based on the predicted CBF₁ gene sequence (XM_008440940). These sequences were used as the templates in blast searches against the melon genome databases (https://melonomics.net), and the ATG and TAA codons of the coding regions were determined. Specific primers were designed based on the conserved regions of CBF₁, The resulting cDNA was employed as a template for PCR reactions using gene-specific primers to amplify the coding regions of CmCBF₁, the expression of CmCBF₁ was carried out in triplicate with different cDNA samples synthesized from tissue collected at each sampling time with PCR amplification.【Result】The CmCBF₁ gene from the cDNA contained an open reading frame of 639 bp, its GenBank accession number was KT737742, which encoded a polypeptide of 212 amino acids residues with a molecular mass of 23.89 kD and a pI of 5.23. The sequence homology comparison showed that the CmCBF₁ had a relatively close evolutionary relationship with Fragariaananassa, FaCmCBF₁, which shared 85 %. RT-PCR analysis showed that the expression of CmCBF₁ was induced by low temperature, including 1℃、3℃ and 5℃, but not expressed at room temperature at 20℃.【Conclusion】There is a negative correlation between the relative expression of transcription factor CmCBF₁ and the incidence of chilling injury of Hami melon fruit, which provides the theoretical basis for studying the mechanism of the incidence of chilling injury of Hami melon fruit during the storage at low temperature.

Key words: Hami melon fruit; CmCBF₁; gene cloning; gene expression

中图分类号:

S652

张婷, 潘俨, 孟新涛, 郑素慧, 徐斌. 哈密瓜果实转录因子CmCBF₁的克隆及表达分析[J]. 新疆农业科学, 2017, 54(10): 1765-1774.

ZHANG Ting, PAN Yan, MENG Xin-tao, ZHENG Su-hui, XU Bin. Cloning and Expression Analysis of a CRT/DRE-binding Factor Gene CmCBF₁ from Hami Melon Fruit[J]. Xinjiang Agricultural Sciences, 2017, 54(10): 1765-1774.

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哈密瓜果实转录因子CmCBF₁的克隆及表达分析

Cloning and Expression Analysis of a CRT/DRE-binding Factor Gene CmCBF₁ from Hami Melon Fruit

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