松墨天牛小热激蛋白基因的克隆、表达谱及对温度胁迫的响应

cDNA cloning and expression profiling of small heat shock protein genes and their response to temperature stress in Monochamus alternatus( Coleoptera: Cerambycidae)

【目的】松墨天牛Monochamus alternatus是我国南方松林中的重要蛀干害虫,也是林业检疫性病害——松材线虫病的主要媒介昆虫,其分布范围较广,对温度的适应性强,本研究旨在初步探讨松墨天牛对温度胁迫适应性的分子机制。【方法】采用RT-PCR与RACE技术克隆松墨天牛小热激蛋白(small heat shock protein,sHSP)基因的全长cDNA,结合生物信息学方法分析小热激蛋白的结构特征;利用qPCR技术测定该基因在松墨天牛不同发育阶段、4龄幼虫不同组织及不同低温和高温胁迫下4龄幼虫中的表达谱,并用geNorm,NormFinder和BestKeeper软件对不同温度下内参基因稳定性进行评价。【结果】获得松墨天牛sHSP基因的cDNA全长序列,命名为MaltHSP21.20(GenBank登录号:MH091811),全长为871 bp,编码187个氨基酸,信号肽预测表明其N末端含有18个氨基酸的信号肽,成熟蛋白预测分子质量为21.20 kD,等电点为8.65。结构域预测符合小热激蛋白家族的特征,共含有10个β折叠片,在α结构域内含有7个β折叠片。进化树分析表明该蛋白氨基酸序列与光肩星天牛Anoplophora glabripennis的sHSP有较高的同源性。不同软件分析得到最稳定的内参基因存在差异,结合3种方法评价得出RPL10最为稳定。MaltHSP21.20在不同发育阶段的松墨天牛体内均有表达,滞育幼虫中表达量最高,卵期及蛹期表达量次之;在4龄幼虫不同组织中均有分布,脂肪体中表达量最高;4龄幼虫MaltHSP21.20对低温诱导无响应,其表达量在35℃时显著上调,45℃处理2和3 h最高,50℃时下降。【结论】RPL10是不同温度胁迫下较稳定的内参基因。相比其他热激蛋白基因,MaltHSP21.20对低温敏感性较低,推测其在幼虫滞育越冬及抵抗高温中发挥重要作用。

【Aim】Monochamus alternatus,an important stem borer in pine forests in southern China,is a major vector of pine wilt disease.It has a wide range of distribution and strong resistance to temperature stress.The study aims to better understand the molecular mechanism of resistance to temperature stress in M.alternatus.【Methods】The full-length cDNA encoding small heat shock protein(sHSP)was cloned from M.alternatus by RT-PCR and RACE techniques.The sequence characteristics of this sHSP were analyzed by bioinformatics methods.The expression patterns of this gene in different developmental stages,various tissues of the 4th instar larvae and the 4th instar larvae stressed under different low and high temperatures were detected by qPCR,and the stability of the reference genes at different temperatures was analyzed by geNorm,NormFinder and BestKeeper tools.【Results】The complete cDNA of the gene encoding sHSP was obtained from M.alternatus,and named MaltHSP 21.20(GenBank accession no.:MH091811).The complete cDNA(871 bp)encodes a protein of 187 amino acids,with a signal peptide of 18 amino acids in N-terminus,the molecular weight of 21.20 kD and the theoretical pI of 8.65.The domain prediction conforms to the characteristics of the sHSP family,containing 10β-sheets and 7β-sheets in theα-domain.Phylogenetic tree analysis showed that the sHSP sequence shares high homology with sHSP of Anoplophora glabripennis.The most stable reference gene was different identified by different methods,and assessed by three methods the most stable reference gene was RPL 10.MaltHSP 21.20 was expressed in all developmental stages of M.alternatus,with the highest expression level in diapause larvae,and moderate in egg and pupal stages.MaltHSP 21.20 was expressed in different tissues of the 4th instar larvae,with the highest expression level in fat body.The expression of MaltHSP 21.20 in the 4th instar larvae showed no response to low temperature,while was up-regulated significantly at 35℃,reached the maximum after exposure to 45℃for 2 and 3 h,and down-regulated at 50℃.【Conclusion】RPL 10 is a more stable reference gene under different temperature stress.Compared to other heat shock protein genes,MaltHSP 21.20 is less sensitive to low temperature and presumably plays an important role in larval diapause during overwintering and the resistance to high temperature.