虫酰肼对斜纹夜蛾幼虫多巴脱羧酶基因表达的影响

doi:10.3864/j.issn.0578-1752.2014.08.007

摘要/Abstract

摘要： 【目的】克隆斜纹夜蛾（Spodoptera litura）多巴脱羧酶（dopa decarboxylase，DDC）基因cDNA全长，研究虫酰肼对表皮形成过程中多巴脱羧酶基因表达的影响。【方法】通过RT-PCR和RACE技术克隆得到斜纹夜蛾多巴脱羧酶基因的cDNA全长；通过生物信息学工具对斜纹夜蛾多巴脱羧酶基因的DNA序列及其编码的蛋白质序列进行分析；采用饲料浸毒法测定虫酰肼对斜纹夜蛾不同龄期幼虫的毒力；利用荧光实时定量PCR检测用亚致死剂量虫酰肼处理的斜纹夜蛾幼虫DDC基因的表达水平。【结果】克隆了斜纹夜蛾多巴脱羧酶基因的cDNA全长，开放阅读框为1 263 bp，编码420个氨基酸，5′端非编码区长105 bp，3′端非编码区长79 bp。克隆得到的cDNA全长提交至GenBank，登录号为KF620492。多重序列比对及系统进化树显示斜纹夜蛾DDC基因推导的氨基酸序列与鳞翅目昆虫的DDC序列具有很高的相似性。利用SWISS-MODEL在线工具进行同源建模，成功预测斜纹夜蛾DDC三维结构，其与果蝇属（Drosophila）的多巴脱羧酶空间结构具有很高的相似性，功能结构域具有良好的保守性。测定虫酰肼对斜纹夜蛾3、4、5和6龄幼虫的毒力，其LC50分别为33.32、40.28、71.20和71.97 mg•L-1。DDC转录表达结果显示，用虫酰肼分别处理12—48 h，4个亚致死剂量（7.50、16.24、28.34和45.63 mg•L-1）诱导的DDC表达水平均显著高于对照，随着处理时间的延长激活作用呈现出先上升后下降的趋势，其中16.24 mg•L-1亚致死剂量在24、36和48 h诱导表达水平最高，各处理36 h表达水平最高。处理60 h，4个亚致死剂量的表达水平均显著低于对照。这一结果说明虫酰肼进入虫体后先启动基因表达，而后抑制表达，影响昆虫正常蜕皮。【结论】克隆得到斜纹夜蛾多巴脱羧酶基因cDNA全长，虫酰肼干扰昆虫幼虫蜕皮与其影响表皮形成过程中多巴脱羧酶基因的表达有关。

关键词: 斜纹夜蛾 , 虫酰肼 , 多巴脱羧酶 , 基因克隆 , 实时定量PCR

Abstract: 【Objective】 The objective of this study is to clone the full length cDNA of dopa decarboxylase (DDC) gene from 5th instar larvae of Spodoptera litura Fabricius and to investigate the effects of tebufenozide on mRNA expression of DDC gene in the process of cuticular formation. 【Method】 RT-PCR and RACE techniques were used to clone the complete cDNA of DDC gene from S. litura. Bioinformatics tools were applied to analyze both the DNA sequence and protein sequence. Toxicity of tebufenozide to different instar larvae of S. litura was determined by artificial diet dipping method. The mRNA expression of the gene in 5th instar larvae of S. litura which were continuously treated with tebufenozide for different lengths of time were investigated using real time quantitative PCR. 【Result】 The complete DDC gene from 5th instar larvae of S. litura was cloned. Open reading frame (ORF) of DDC was 1 263 bp in length, encoding 420 amino acids. The 5′ end and 3′ end non-coding regions were 105 and 79 bp in length. The sequence was deposited in GenBank database with accession number of KF620492. Homology and phylogenetic analysis indicated that the DDC of S. litura had very high similarity with that of Lepidoptera. The DDC three-dimensional structure was successfully predicted by means of SWISS-MODEL online tools. The structure was similar to the DDC structure of Drosophila. The function structure domain had good conservative property. The results showed that the toxicity of 3rd, 4th, 5th and 6th instar larvae’s LC50 were 33.32, 40.28, 71.20 and 71.97 mg?L-1, respectively. The DDC gene mRNA expression was significantly higher than CK when the larvae were treated with four tebufenozide sublethal doses (7.50, 16.24, 28.34, 45.63 mg?L-1) for 12, 24, 36 and 48 h, respectively. The activation rates raised and then declined with tebufenozide-treated time increasing. The expression of the larvae treated with 16.24 mg?L-1 sublethal doses was the highest after 24, 36 and 48 h tebufenozide-treated time. Meanwhile, the four tebufenozide sublethal doses had the highest expressions after 36 h. The DDC gene mRNA expression had a sharp decrease and was significantly lower than CK after 60 h treated time. The tebufenozide activated the DDC gene expression and then inhibited it. 【Conclusion】 DDC gene was cloned and characterized from S. litura. The interference in the formation of new larval cuticle by tebufenozide is closely related to the effects on DDC gene mRNA expression by tebufenozide.

Key words: Spodoptera litura , tebufenozide , dopa decarboxylase (DDC) , gene cloning , real time quantitative PCR

张文慧, 侯海霞, 杨琼, 王淑会, 毛旭连, 刘永杰. 虫酰肼对斜纹夜蛾幼虫多巴脱羧酶基因表达的影响[J]. 中国农业科学, 2014, 47(8): 1512-1521.

ZHANG Wen-Hui, HOU Hai-Xia, YANG Qiong, WANG Shu-Hui, MAO Xu-Lian, LIU Yong-Jie. Effects of Tebufenozide on mRNA Expression of Dopa Decarboxylase Gene in Larvae of Spodoptera litura Fabricius[J]. Scientia Agricultura Sinica, 2014, 47(8): 1512-1521.

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