›› 2014, Vol. 57 ›› Issue (11): 1253-1264.

• 研究论文 •    下一篇

温度胁迫下棉花粉蚧热激蛋白基因的表达分析

陈芳, 陆永跃*   

  1. (华南农业大学昆虫学系, 广州510642)
  • 出版日期:2014-11-20 发布日期:2014-11-20
  • 作者简介:陈芳, 女, 1989年11月生, 江西鹰潭人, 硕士研究生, 研究方向为昆虫分子生态学, E-mail: chenfang008@foxmail.com

Expression analysis of heat shock protein genes in Phenacoccus solenopsis (Hemiptera: Pseudococcidae) under temperature stress

CHEN Fang, LU Yong-Yue*   

  1. (Department of Entomology, South China Agricultural University, Guangzhou 510642, China)
  • Online:2014-11-20 Published:2014-11-20

摘要: 【目的】为了研究热激蛋白 Hsp70, Hsp70-4和Hsp90在棉花粉蚧Phenacoccus solenopsis抵抗逆温中的作用。【方法】在测序棉花粉蚧转录组的基础上,分析了该虫热激蛋白Hsp70基因家族的2个序列[Pshsp70(GenBank登录号为KJ909505)和Pshsp70-4(GenBank登录号为KJ909506)]和Hsp90基因家族的1个序列,[Pshsp90(GenBank登录号为KJ909507)],采用实时荧光定量 PCR(RT-qPCR)检测了在不同温度(18和32℃恒温, 37, 39, 41, 43和45℃热激1 h 后26℃恢复1 h)下棉花粉蚧不同发育阶段(2龄若虫、3龄若虫、雌成虫)3种热激蛋白基因的表达量。【结果】Pshsp70 cDNA序列包含1 923 bp的开放阅读框,编码641个氨基酸,理论分子量和等电点分别为70.9 kDa和5.65; Pshsp70-4 cDNA序列包含1 962 bp的开放阅读框,编码654个氨基酸,理论分子量和等电点分别为71.8 kDa和5.38;Pshsp90 cDNA序列包含2 172 bp的开放阅读框,编码724个氨基酸,理论分子量和等电点分别为83.5 kDa和4.93。Pshsp70 和Pshsp70-4均含有Hsp70基因家族高度保守的基序,Pshsp70编码的氨基酸序列与烟粉虱Bemisia tabaci和家蚕Bombyx mori等昆虫的Hsp70 的氨基酸序列一致性为 85%;Pshsp70-4编码的氨基酸序列与白蜡蚧Ericerus pela和点蜂缘蝽Riptortus pedestris等昆虫的Hsp70的氨基酸序列一致性高达95%;Pshsp90也含有Hsp90基因家族高度保守的基序,Pshsp90编码的氨基酸序列与赤拟谷盗Tribolium castaneum和东亚小花蝽Orius sauteri等昆虫的Hsp90 的氨基酸序列一致性为 87%。热激蛋白基因表达量分析结果表明,在18℃恒温条件下,粉蚧2龄若虫的3个PsHsps基因的mRNA相对表达量均比对照(26℃)低,在32℃恒温条件下,各龄期的Hsp70基因的相对表达量均显著高于对照。在37~45℃下热激1 h并在26℃下恢复1 h,棉花粉蚧3个龄期的3个热激蛋白PsHsps基因的相对表达量随温度的升高总体呈增加趋势,相关性分析表明,除Pshsp70-4在雌成虫中的表达量与热胁迫温度的相关系数为0.225外,各龄期中3个基因的表达量与温度的相关系数均大于0.6,显著相关;43℃和45℃胁迫下,各龄期的3个热激蛋白基因相对表达量均显著高于对照组(P<0.05)。【结论】棉花粉蚧热激蛋白基因的表达与温度呈正相关,在该虫应对高温中起着重要作用。

关键词:  , 棉花粉蚧; 热激蛋白; 温度胁迫; 序列分析; mRNA相对表达量; 实时荧光定量PCR

Abstract: 【Aim】 This study aims to study the roles of heat shock protein in the adaptation to temperature stress in Phenacoccus solenopsis. 【Methods】 Based on the successful transcriptome sequencing in P. solenopsis, we analyzed two sequences of heat shock protein 70 gene family [PsHsp70 (GenBank accession no. KJ909505) and Pshsp70-4 (GenBank accession no. KJ909506)] and one sequence of heat shock protein 90 gene family [Pshsp90 (GenBank accession no. KJ909507)], and used comparative quantitative real-time PCR (RT-qPCR) to analyze the expression of genes Pshsp70, Pshsp70-4 and Pshsp90 at different developmental stages (2nd instar nymph, 3rd instar nymph and female adult) of P. solenopsis in response to different temperatures (constant temperature 18 and 32℃, and temporary heat shock for 1 h at 37, 39, 41, 43 and 45℃ and then recovery for 1 h at 26℃) were detected. 【Results】 Sequence analysis showed that the Pshsp70 cDNA contains a 1 923 bp open reading frame, encoding a polypeptide of 641 amino acids with an estimated molecular mass of 70.9 kDa and an estimated isoelectric piont (pI) of 5.65. The Pshsp70-4 cDNA contains a 1 962 bp open reading frame, encoding a polypeptide of 654 amino acids with an estimated molecular mass of 71.8 kDa and an estimated isoelectric piont (pI) of 5.38. The Pshsp90 cDNA contains a 2 172 bp open reading frame, encoding a polypeptide of 724 amino acids with an estimated molecular mass of 83.5 kDa and an estimated isoelectric piont (pI) of 4.93. Both Pshsp70 and Pshsp70-4 contain the highly conserved functional motifs of the Hsp70 gene family. PsHsp70 shares 85% amino acid sequence identities with the Hsp70 proteins from Bemisia tabaci and Bombyx mori, while PsHsp70-4 shares 95% amino acid sequence identities with the Hsp70 proteins from Ericerus pela and Riptortus pedestris. Pshsp90 contains the highly conserved functional motifs of the Hsp90 gene family, and PsHsp90 shares 87% amino acid sequence identities with the Hsp90 proteins from Tribolium castaneum and Orius sauteri. The expression analysis result showed that the mRNA expression levels of the three Hsp genes in the 2nd instar nymphs were down-regulated at constant temperature 18℃, as compared with those at 26℃(the control), while at 32℃, the mRNA expression level of Pshsp70 was significantly higher than the control group in these three developmental stages. Under heat shock at 37-45℃ for 1 h and then recovery for 1 h, the relative expression levels of the three Hsp genes increased with the increase of temperature in the three different developmental stages. The correlation analysis suggested that the correlation coefficients between the expression level of the three Hsp genes in all developmental stages and the heat stress temperature were greater than 0.6, except that of Pshsp70-4 in female adult (0.225). The expression levels of the three Hsp genes in all tested stages under heat shock at 43℃ and 45℃ for 1 h and then recovery for 1 h were significantly higher than the control group. 【Conclusion】 The expression of heat shock protein genes in P. solenopsis is positively correlated with temperature, and PsHsps may play an important role in high temperature tolerance in P. solenopsis.

Key words: Phenacoccus solenopsis, heat shock protein (Hsp), temperature stress, sequence analysis, mRNA expression level, quantitative real-time PCR