哈密瓜果实转录因子CmCBF_1的克隆及表达分析  

Cloning and Expression Analysis of a CRT/DRE-binding Factor Gene CmCBF_1 from Hami Melon Fruit

作　　者：张婷 潘俨 孟新涛 郑素慧 徐斌 ZHANG Ting PAN Yan MENG Xin - tao ZHENG Su -hui XU Bin(Research Institute of Agricultural Products Storage and Processing, Xinjiang Academy of Agricultural Sciences, Urumqi 830091, China)

机构地区：[1]新疆农业科院农产品贮藏加工研究所,乌鲁木齐830091

出　　处：《新疆农业科学》2017年第10期1765-1774,共10页Xinjiang Agricultural Sciences

基　　金：新疆维吾尔自治区自然科学基金项目"哈密瓜抗冷途径中CBF转录因子基因的克隆与表达"(2014211B026)~~

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CmCBF_1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c DNA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CmCBF_1,该基因序列为639 bp(Gen Bank登录号为KT737742),编码212个氨基酸,蛋白质分子量为23.89 k Da,等电点为5.23;同源性分析表明,哈密瓜果实转录因子CmCBF_1与草莓Fa CBF1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CmCBF_1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)。[ Objective] In order to make clear the regulatory function of 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. 3v Hami melon postharvest fruit was selected as materials. The primers used to am-plify 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 databas-es （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 tem-plate 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 CttiCS.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 tempera-ture, 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 ch il l-ing injury of Hami melon fruit, which provides the theoretical basis for studying the mechanism of the inci-dence of chilling injury of Hami melon fruit during the storage at low temperature.

关 键 词：哈密瓜 CM CBF1 克隆 表达 

分 类 号：S652[农业科学—果树学]