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【目的】近年来,大螟Sesamia inferens(Walker)对水稻的为害逐渐加重并成为水稻的重要害虫之一。随着全球气候变暖,大螟的分布区域也在逐渐向北延伸。HSP70家族作为分子伴侣参与生物生长发育并对外界刺激产生响应,对生物功能蛋白质的正确折叠及其转运有着重要意义。本研究旨在明确HSP70家族HSC70基因在大螟不同组织、不同发育阶段以及低温胁迫下的表达差异,初步探讨大螟对环境适应的分子机理。【方法】应用RT-PCR及RACE技术从大螟5龄幼虫中克隆得到HSC70基因;进行基因组验证,得到其基因组序列,分析内含子的位置及大小;应用实时定量PCR技术分析大螟HSC70基因的表达模式。【结果】大螟HSC70基因长2 160 bp,命名为Sihsc70(GenBank登录号:KJ639908),开放阅读框长1 962 bp,编码653个氨基酸,推测分子量为71.6 kDa。其氨基酸序列中含有3个HSP70家族保守序列,在C-末端存在细胞质定位信号,说明大螟HSC70是细胞质热激蛋白家族成员。大螟HSC70基因组序列长度为3 522 bp(GenBank登录号:KJ639909),含有2个内含子,长度分别为685 bp(位于编码区上游)和803 bp(位于编码区内)。在大螟5龄幼虫的不同组织中Sihsc70表达量差异不显著(P>0.05),其中在中肠、后肠和体壁中的表达量较高,在唾腺中的表达量最低;在大螟不同发育阶段中,Sihsc70的表达量在雌成虫最低,较高的3个阶段依次为卵、2龄幼虫和5龄幼虫,分别为雌成虫表达量的6.33,3.21和1.86倍;相对于对照组(27℃),低温胁迫对大螟5龄幼虫HSC70基因表达的影响差异不显著(P>0.05)。【结论】结果说明,大螟HSC70基因在不同发育阶段和幼虫不同组织中具有不同的表达水平,而低温胁迫不能诱导该基因大量表达。
【Objective】 In recent years, the damage of Sesamia inferens (Walker) to rice has gradually increased and become one of the important pests in rice. With the global warming, the distribution area of Sesamia inferers also gradually extends northward. The HSP70 family participates in the biological growth and development as a chaperone and responds to external stimuli, which is of great significance for the correct folding and transport of bio-functional proteins. The purpose of this study was to determine the expression difference of HSP70 family HSC70 gene in different tissues, different developmental stages and low temperature stress of Sesamia inferens. The molecular mechanism of adaptation of Sesamia inferens to environment was discussed. 【Method】 The HSC70 gene was cloned from 5th instar larvae of Sesamia inferens by RT-PCR and RACE techniques. Genomic sequence analysis was carried out to obtain the HSC70 gene. The position and size of introns were analyzed. Real-time PCR was used to analyze the HSC70 gene The expression pattern. 【Result】 The results showed that the HSC70 gene of Sesamia inferens is 2 160 bp in length and named Sihsc70 (GenBank accession number: KJ639908). The open reading frame of HSC70 gene is 1 962 bp in length and encodes a protein of 653 amino acids with a predicted molecular weight of 71.6 kDa. Its amino acid sequence contains three HSP70 family conserved sequences, there is a cytoplasmic localization signal at the C-terminal, indicating that Stem Borer HSC70 is a member of the cytoplasmic heat shock protein family. The sequence length of HSC70 was 3 522 bp (GenBank accession number: KJ639909), containing 2 introns, with a length of 685 bp (upstream of the coding region) and 803 bp (located within the coding region), respectively. The expression of Sihsc70 in different tissues of 5th instar larvae of Sesamia inferens showed no significant difference (P> 0.05). Among them, the expression of Sihsc70 was higher in midgut, hindgut and body wall and lowest in salivary glands; In the different developmental stages, Sihsc70 expression was the lowest in female adults, followed by eggs, 2nd instar larvae and 5th instar larvae, respectively, which were 6.33, 3.21 and 1.86 times higher than the control (27 ℃), cold stress on the fifth instar larvae HSC70 gene expression difference was not significant (P> 0.05). 【Conclusion】 The results show that the HSC70 gene of Sesamia inferens has different expression levels in different developmental stages and different tissues of larvae, while cold stress can not induce the gene expression in large quantities.