黄粉虫热休克蛋白70基因的克隆、序列分析与表达（英文）

为给黄粉虫Tenebrio molitor抗逆机理研究提供理论依据， 本研究采用PCR和RACE法从黄粉虫幼虫中克隆出一个热休克蛋白70基因Tmhsp70， 并运用半定量RT-PCR法检测其在黄粉虫不同发育阶段的mRNA表达水平。结果表明： 克隆出的Tmhsp70 序列全长2 282 bp， 具有一个富含A的115 bp 5′ 非翻译区和一个1 935 bp的开放阅读框及一个富含A、 T的232 bp 3′-非翻译区。5′-非翻译区含有7个热休克元件nGAAn， 3′-非翻译区末端有长22 bp的Poly(A)尾。Tmhsp70编码的黄粉虫热休克蛋白（TmHSP70）具有3个典型的HSP70特征基序（IDLGTTYS, IFDLGGGTFDVSIL和IVLVGGSTRIPKIQQ）和1个胞质HSP70末端特征基序（EEVD）， 无信号肽和跨膜区域， 包含2个主要的结构域， 即： N-端42 kDa的高度保守ATPase功能域和C-端18 kDa的保守多肽结合功能域。ATPase功能域的三级结构由2个大球形亚功能域组成， 具有1个核苷酸结合中心； 多肽结合功能域形成1个双层4股β-折叠片样的三明治结构和2个α-螺旋， 内含1个多肽结合通道。此外， 黄粉虫Tmhsp70 mRNA的表达具有热激诱导和发育调控的特征。半定量RT-PCR分析表明， 42℃热激1 h的黄粉虫各发育阶段Tmhsp70 mRNA的表达量上升了1.4~26.9倍。25℃下1日龄黄粉虫蛹中的Tmhsp70 mRNA 表达量要高于其余各发育阶段的累积表达量； 42℃热激1 h 后90日龄幼虫中的Tmhsp70 mRNA 表达量最丰富， 既高于30日龄和60日龄幼虫中的累积表达量， 也高于15日龄和30日龄成虫中的累积表达量。这些结果为进一步研究黄粉虫热休克蛋白的结构、 功能和表达调控及其与抗逆性的关系奠定了基础。

Cloning, sequence analysis and expression profiling of a heat shock protein 70 gene in Tenebrio molitor (Coleoptera: Tenebrionidae)

In order to study the mechanism of stress-resistance, a heat shock protein gene hsp70 (named as Tmhsp70), was cloned from the larvae of Tenebrio molitor by PCR and RACE method, and the mRNA levels in developmental stages were detected by using semi-quantitative RT-PCR as well. The results showed the full sequence of Tmhsp70 cloned is 2 282 bp in length containing a 115 bp 5′ untranslated region (5′ UTR) rich in adenine, a 1 935 bp open reading frame and a 232 bp 3′ untranslated region (3′ UTR) rich in adenine and thymine. It also has seven repeats of the heat shock element nGAAn in its 5′ UTR and a 22 bp Poly (A) tail in the 3′ UTR. The deduced heat shock protein named as TmHSP70 contains three signature motifs of HSP70, i.e., IDLGTTYS, IFDLGGGTFDVSIL and IVLVGGSTRIPKIQQ as well as the terminal EEVD motif which is characteristic to cytoplasmic HSP70s. TmHSP70 has neither a signal peptide nor a transmembrane domain. It contains two main functional domains: a 42 kDa highly conserved N-terminal ATPase domain and a 18 kDa conserved C-terminal peptide-binding domain. The tertiary structure of ATPase domain is composed of two large globular subdomains and contains a nucleotide-binding core. Tertiary structure of the peptide-binding domain forms a sandwich of 2 four-stranded β-sheets and two α-helices, and includes a peptide-binding cavity. Furthermore, the expression of Tmhsp70 mRNA in T. molitor was characterized by heat-inducible and developmental regulation feature. The overall increase in the levels of Tmhsp70 mRNA in different life stages when the larvae were exposed to 42℃ for 1 h, ranged from 1.4- to 26.9- fold on the basis of semi-quantitative RT-PCR analysis. At 25℃ Tmhsp70 mRNA expressed in 1-day old pupae was higher than that accumulated in other developmental stages, and after exposure to 42℃ for 1 h, Tmhsp70 mRNA expressed in 90-day old larvae became the most abundant, and was not only higher than that accumulated in 30- and 60-day old larvae but also higher than that accumulated in 15- and 30-day old adults. The results form a basis for further research on structure, function and expression regulation of HSPs from T. molitor as well as the relationship between HSPs and stress-resistance in the beetle.