黄粉虫热休克蛋白70基因的克隆、序列分析与表达（英文）

›› 2013, Vol. 56 ›› Issue (5): 475-485.doi:

黄粉虫热休克蛋白70基因的克隆、序列分析与表达（英文）

黄琼^1,*，胡杰²，孙灵¹，王勤³

（1. 四川农业大学林学院森林保护学省级重点实验室，四川雅安 625014; 2. 四川农业大学经济管理学院，成都 611130;
3. 四川农业大学生命科学理学院生物系，四川雅安 625014）

出版日期:2013-05-20 发布日期:2013-05-20

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

HUANG Qiong^1,*, HU Jie², SUN Lin¹, WANG Qin³

(1. Provincial Key Laboratory of Forest Protection, College of Forestry, Sichuan Agricultural University, Ya’an,
Sichuan 625014, China; 2. College of Economics and Management, Sichuan Agricultural University, Chengdu 611130, China;
3. Department of Biology, College of Life and Basic Sciences, Sichuan Agricultural University, Ya’an, Sichuan 625014, China)

Online:2013-05-20 Published:2013-05-20

摘要/Abstract

摘要： 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。

关键词: 黄粉虫, 热休克蛋白, 基因克隆, 生物信息学, mRNA表达, 热激

Abstract: 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.

Key words: Tenebrio molitor, heat shock protein (HSP), gene cloning, bioinformatics, mRNA expression, heat shock

黄琼, 胡杰, 孙灵, 王勤. 黄粉虫热休克蛋白70基因的克隆、序列分析与表达（英文）[J]. , 2013, 56(5): 475-485.

HUANG Qiong, HU Jie, SUN Lin, WANG Qin. Cloning, sequence analysis and expression profiling of a heat shock protein 70 gene in Tenebrio molitor (Coleoptera: Tenebrionidae) (In English)[J]. , 2013, 56(5): 475-485.

0
/ / 推荐

导出引用管理器 EndNote|Reference Manager|ProCite|BibTeX|RefWorks

链接本文: http://www.insect.org.cn/CN/

http://www.insect.org.cn/CN/Y2013/V56/I5/475

[1]	马荣琴, 王林玲, 衡惠, 周泽扬, 李治. 意大利蜜蜂铁蛋白基因AmFer3HCH的克隆及其在温度胁迫下的应答[J]. , 2018, 61(9): 1029-1039.
[2]	李慧, 何玄玉, 陶蓉, 陈宏建, 龚馨玥, 李寿银, 郝德君. 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[J]. , 2018, 61(7): 749-760.
[3]	单艳敏, 张玉, 霍志家, 庞保平, 孙学涛. 沙葱萤叶甲丝氨酸蛋白酶基因GdSP的克隆及对温度胁迫的响应[J]. , 2018, 61(7): 761-770.
[4]	李鹏燕, 刘艳萍, 王思威, 孙海滨, 柏建山, 彭刚, 龚雪海. 荔枝蒂蛀虫信息素结合蛋白基因序列及表达分析[J]. , 2018, 61(5): 527-536.
[5]	司品法, 周琼, 崔中翌 . 柑橘大实蝇气味结合蛋白基因BminOBP25的克隆、原核表达及组织表达分析[J]. , 2018, 61(5): 537-545.
[6]	岳金金, 马宇驰, 刘全, 姜莉莉, 费东亮, 张鹤, 孙莉, 李明, 马鸣潇. 中华蜜蜂半乳糖凝集素AcGalectin的表达、纯化及性质分析[J]. , 2018, 61(5): 546-554.
[7]	李小龙, 熊晓莉, 李宁. 基于极端顶点混料设计优化黄粉虫秸秆饲料配方[J]. , 2018, 61(5): 596-603.
[8]	程杏安, 林贤伟, 蒋旭红, 刘展眉, 钟国华, 胡美英. 草地贪夜蛾组织蛋白酶L的基因克隆、原核表达及多克隆抗体制备[J]. , 2018, 61(4): 410-422.
[9]	郗学鹏, 魏伟, 张卫星, 王红芳, 胥保华. 中华蜜蜂转录因子AP-1基因的克隆及特性分析[J]. , 2018, 61(4): 423-431.
[10]	陈龙, 谭瑶, 周晓榕, 庞保平, 格希格都仁, 孙志鹏. 沙葱萤叶甲海藻糖酶基因GdTre1的克隆、分子特性和表达分析[J]. , 2018, 61(3): 271-281.
[11]	李鸿波, 戴长庚, 张昌容, 何永福, 胡阳. 粘虫过氧化氢酶基因的克隆与表达分析[J]. , 2018, 61(2): 178-187.
[12]	宋帅葆, 艾上杰, 关怀, 韩谦. 冈比亚按蚊犬尿氨酸甲酰胺酶抑制剂的虚拟筛选[J]. , 2018, 61(1): 68-78.
[13]	谭永安, 赵旭东, 肖留斌, 孙洋, 赵静, 柏立新, 郝德君. 绿盲蝽核受体基因AlE75D的克隆和性质分析[J]. , 2017, 60(9): 984-993.
[14]	伍绍龙, 周志成, 彭曙光, 李玉辉, 李保平, 孟玲. 管氏肿腿蜂母子亲缘关系对抚育行为的影响[J]. , 2017, 60(9): 1041-1045.
[15]	谭瑶, 张玉, 霍志家, 周晓榕, 单艳敏, 庞保平. 沙葱萤叶甲热激蛋白基因GdHsp70的克隆与表达模式分析[J]. , 2017, 60(8): 865-875.