昆虫学报 ›› 2021, Vol. 64 ›› Issue (12): 1407-1416.doi: 10.16380/j.kcxb.2021.12.006

• 研究论文 • 上一篇    下一篇

菜粉蝶热激蛋白70(HSP70)基因的分子特性及其对杀虫剂胁迫的响应

丁朝阳#, 赵乐#, 刘苏*, 李茂业   

  1. (安徽农业大学植物保护学院, 作物有害生物综合治理安徽省重点实验室, 合肥230036)
  • 出版日期:2021-12-20 发布日期:2021-11-26

Molecular characterization of heat shock protein 70 (HSP70) genes from Pieris rapae (Lepidoptera: Pieridae) and their responses to insecticide stress

DING Chao-Yang#, ZHAO Le#, LIU Su*, LI Mao-Ye   

  1.  (Anhui Provincial Key Laboratory of Integrated Pest Management on Crops, School of Plant Protection, Anhui Agricultural University, Hefei 230036, China)
  • Online:2021-12-20 Published:2021-11-26

摘要:

【目的】明确菜粉蝶Pieris rapae热激蛋白70(HSP70)基因的分子特性及其对杀虫剂胁迫的响应,为探索菜粉蝶HSP70基因在抵御杀虫剂胁迫中的功能提供前期基础。【方法】利用同源检索方法,从菜粉蝶转录组数据中鉴定HSP70基因;使用生物信息学方法分析HSP70基因的分子特性;采用实时荧光定量PCR技术分析HSP70基因在菜粉蝶不同发育阶段(2-5龄幼虫、蛹和雌雄成虫)、4龄幼虫不同组织(中肠、马氏管、脂肪体和体壁)以及LD20剂量高效氯氟氰菊酯(0.12 ng/μL)和氯虫苯甲酰胺(1.04 ng/μL)胁迫下4龄幼虫体内的表达谱。【结果】从菜粉蝶转录组中鉴定了3个HSP70基因(PrHsp70-1, PrHsp70-2和PrHsp70-3) (GenBank登录号分别为MW691114, MW691115和MW691116),它们分别编码628, 630和653个氨基酸,分子量分别为68.7, 69.2和71.7 kD。生物信息学分析结果表明,3个PrHSP70蛋白均为胞质蛋白,且均含有HSP70家族的特征序列。PrHsp70-1和PrHsp70-2无内含子,而PrHsp70-3含有1个内含子。随着幼虫龄期的增加,PrHsp70-1和PrHsp70-2的表达量也不断上调,但在蛹期和成虫期下调;PrHsp70-3在供试的不同发育阶段样本中的表达量无显著差异。PrHsp70-1高量表达于幼虫脂肪体,PrHsp70-2高量表达于幼虫中肠,PrHsp70-3在幼虫体壁和脂肪体中表达量均较高。LD20剂量高效氯氟氰菊酯和氯虫苯甲酰胺均能诱导3个PrHsp70基因显著上调表达,但不同基因的响应速度有差异;此外,LD20剂量高效氯氟氰菊酯处理24 h后,PrHsp70-3显著下调表达。【结论】PrHsp70基因可能在菜粉蝶生长发育以及抵御杀虫剂胁迫中均有重要作用。

关键词: 菜粉蝶, 热激蛋白, 序列特征, 表达模式, 高效氯氟氰菊酯, 氯虫苯甲酰胺

Abstract:  【Aim】 This study aims to determine the molecular characteristics of heat shock protein 70 (HSP70) genes in the small white butterfly, Pieris rapae and their response to insecticide stress, thus to pave the way for exploring the role of HSP70s in defense against insecticides in P. rapae. 【Methods】 Homology-basedsearchmethodwasusedtoidentifyHSP70 genes from the transcriptome dataset of P. rapae. Bioinformatic programs were used to analyze the molecular characteristics of these HSP70 genes. Real-time quantitative PCR was performed to determine the expression profiles of HSP70 genes at different developmental stages (2nd-5th instar larvae, pupae, and female and male adults), in different tissues of the 4th instar larvae (midgut, Malpighian tubules, fat body and integument) of P. rapae, and in the 4th instar larvae exposed to LD20 of lambda-cyhalothrin (0.12 ng/μL) and chlorantraniliprole (1.04 ng/μL). 【Results】 Three HSP70 genes (PrHsp70-1, PrHsp70-2 and PrHsp70-3 with the GenBank accession numbers of MW691114, MW691115 and MW691116, respectively) were identified from the transcriptome of P. rapae. PrHSP70 proteins encoded by PrHsp70-1, PrHsp70-2 and PrHsp70-3 consist of 628, 630 and 653 amino acid residues, respectively, and have the molecular masses of 68.7, 69.2 and 71.7 kD, respectively. The results of bioinformatic analysis indicated that all of the three PrHSP70 proteins are localized within the cytosol and have the signature motifs of HSP70 family. PrHsp70-1 and PrHsp70-2 are intronless, whereas PrHsp70-3 contains an intron. The expression levels of PrHsp70-1 and PrHsp70-2 were upregulated along with the increase of larval instars and downregulated during pupal and adult stages. By contrast, the expression levels of PrHsp70-3 in P. rapae among different developmental stages showed no significant difference. PrHsp70-1 and PrHsp70-2 were highly expressed in larval fat body and midgut, respectively, while PrHsp70-3 displayed higher transcription level in both larval integument and fat body. Treatment with LD20 of lambda-cyhalothrin and chlorantraniliprole caused significant upregulation of the three PrHsp70 genes, although their responses varied with time. Moreover, the transcription level of PrHsp70-3 was significantly downregulated at 24 h post exposure to LD20 of lambda-cyhalothrin. 【Conclusion】 PrHsp70 genes may play essential roles in the growth and development and insecticide defense in P. rapae.

Key words: Pieris rapae, heat shock protein, sequence characteristics, expression pattern, lambda-cyhalothrin, chlorantraniliprole