桑树热激转录因子(Hsf)基因家族克隆及MnHsfA1基因表达模式分析

Cloning of heat shock transcription factor(Hsf)gene family from mulberry and analysis of MnHsfA1 gene expression pattern

【目的】克隆桑树热激转录因子(Hsf)基因家族成员,并分析MnHsfA1基因表达模式,为深入研究该家族在桑树热应激响应中的调控机制及桑树育种提供理论参考。【方法】以川桑幼苗为试验材料,克隆其MnHsfs基因,通过多序列比对、构建系统发育进化树和MEME保守基序在线预测分析,对MnHsfs蛋白的理化性质、保守结构域、信号肽、跨膜结构域、亚细胞定位和启动子顺式作用元件进行预测,利用实时荧光定量PCR检测MnHsfA1基因在高温和干旱胁迫处理下的表达模式。【结果】从桑树中克隆到14个MnHsfs基因的全长编码区(CDS)序列,分别为MnHsfA4a、MnHsfA4b、MnHsfA2、MnHsfA5、MnHsfA6b-1、MnHsfA6b-2、MnHsfB4-2、MnHsfA8、MnHsfB3、MnHsfB2b、MnHsfC1、MnHsfB2a、MnHsfB4-1和MnHsfA1,长度分别为1239、1278、1404、1431、1101、1140、1185、1197、711、996、1029、936、900和1437 bp,分别编码412、425、467、476、366、379、394、398、236、331、342、312、299和478个氨基酸残基,大部分MnHsfs蛋白是一个亲水、不稳定的酸性蛋白。MnHsfs蛋白包括结合功能域(DBD)、寡聚化功能域(OD)、AHA和KLFGV等结构域,可分为A、B和C类。MnHsfA1与苹果、菠菜和拟南芥的HsfA1相似性最高;与对照相比,MnHsfA1基因相对表达量在高温和干旱胁迫处理下均显著上调(P<0.05)。【结论】MnHsfs基因具有较高的遗传保守性,高温和干旱胁迫诱导MnHsfA1基因高表达,且MnHsfA1基因的启动子顺式作用元件中含有脱落酸、茉莉酸甲酯、赤霉素等激素响应元件以及与各种非生物胁迫相关的转录因子,推测其同时调节多种非生物胁迫。

【Objective】This paper cloned the heat shock transcription factor(Hsf)gene family members in mulberry,analyzed expression pattern of the MnHsfA1 genes to provide theoretical reference for in-depth research on the regulatory mechanism of this family in mulberry heat stress response and mulberry breeding.【Method】Using seedlings of Morus notabilis as experimental materials,the MnHsfs genes were cloned.And the physicochemical properties,conserved domains,signal peptides,transmembrane domains,subcellular localization,and promoter cis acting elements of MnHsfs proteins were predicted by multiple sequence alignment,phylogenetic tree construction,and MEME conserved motif.The expression pattern of MnHsfA1 genes under high temperature and drought stress were analyzed using real-time fluorescence quantitative PCR.【Result】Fourteen full-length coding sequences of MnHsfs genes were cloned from mulberry trees,including MnHsfA4a,MnHsfA4b,MnHsfA2,MnHsfA5,MnHsfA6b-1,MnHsfA6b-2,MnHsfB4-2,MnHsfA8,MnHsfB3,MnHsfB2b,MnHsfC1,MnHsfB2a,MnHsfB4-1 and MnHsfA1.Their open reading frame lengths were 1239,1278,1404,1431,1101,1140,1185,1197,711,996,1029,936,900 and 1437 bp and encoded 412,425,467,476,366,379,394,398,236,331,342,312,299 and 478 amino acids residues respectively.And most of MnHsfs proteins were hydrophilic and unstable acidic proteins.MnHsfs protein included domains such as function domain(DBD),oligomerization function domain(OD),AHA,and KLFGV,and were divided into class A,class B,and class C.The similarity between MnHsfA1 and HsfA1 of apple,spinach,and Arabidopsis were the highest.Compared with the control,the expression of MnHsfA1 gene and mRNA transcription level were significantly up regulated under high temperature and drought stress treatments(P<0.05).【Conclusion】MnHsfs genes have relatively high genetic conservatism,and the expression level of MnHsfA1 gene is up regulated under high temperature and drought stress.The cis acting element of MnHsfA1 gene promoter contains hormone responsive elements such as abscisic acid,methyl jasmonate,gibberellin,as well as transcription factors related to various abiotic stresses.It is speculated that it may regulate multiple abiotic stresses simultaneously.