马铃薯甲虫N-β-丙酰多巴胺水解酶基因的克隆及功能分析

doi:10.16380/j.kcxb.2018.06.002

›› 2018, Vol. 61 ›› Issue (6): 637-645.doi: 10.16380/j.kcxb.2018.06.002

马铃薯甲虫N-β-丙酰多巴胺水解酶基因的克隆及功能分析

杜晓燕^1,2, 付开赟^2,*, 徐晴玉³, 吐尔逊·阿合买提², 丁新华², 何江², 郭文超^4,*

(1. 塔里木大学植物科学学院, 新疆阿拉尔 843300; 2. 新疆农业科学院植物保护研究所, 农业部西北荒漠绿洲作物有害生物综合治理重点实验室, 乌鲁木齐 830091; 3. 南京农业大学植物保护学院, 南京 210095; 4. 新疆农业科学院微生物应用研究所, 乌鲁木齐 830091）

出版日期:2018-06-20 发布日期:2018-06-20

Molecular cloning and functional characterization of N-β-alanyl-dopamine hydroxylase gene in the Colorado potato beetle, Leptinotarsa decemlineata (Coleoptera: Chrysomelidae)

DU Xiao-Yan^1,2, FU Kai-Yun^2,*, XU Qing-Yu³, Tursun AHMAT², DING Xin-Hua², HE Jiang², GUO Wen-Chao^4,*

（1. College of Plant Science,Tarim University, Alar, Xinjiang 843300, China; 2. Key Laboratory of Integrated Pest Management on Crop in Northwestern Oasis, Ministry of Agriculture, Institute of Plant Protection, Xinjiang Academy of Agricultural Sciences, Urumqi 830091, China; 3. College of Plant Protection, Nanjing Agricultural University, Nanjing 210095, China; 4. Institute of Microorganism Application, Xinjiang Academy of Agricultural Sciences, Urumqi 830091, China）

Online:2018-06-20 Published:2018-06-20

摘要/Abstract

摘要： 【目的】通过RNAi技术分析明确对马铃薯甲虫Leptinotarsa decemlineata黑色素形成重要的N-β-丙酰多巴胺(NBAD)水解酶基因的功能。【方法】NBAD水解酶基因通过马铃薯甲虫转录组数据分析和RT-PCR克隆获得，分别利用序列比对和系统发育分析确定该基因的完整性和系统发育；通过qPCR检测其在马铃薯甲虫各发育阶段和4龄幼虫不同组织及成虫精巢和卵巢中的表达量；采用喂食幼虫dsRNA的方法，观察该基因在马铃薯甲虫幼虫的生长发育过程中对体色的影响,并测定保幼激素和蜕皮激素对该基因表达的影响。【结果】克隆得到马铃薯甲虫NBAD水解酶基因，命名为Ldtan(GenBank登录号: KY221866)，其编码蛋白的氨基酸序列与鞘翅目赤拟谷盗Tribolium castaneum和山松大小蠹Dendroctonus ponderosae同源蛋白的氨基酸序列的一致性最高，聚为一支。Ldtan在马铃薯甲虫4龄幼虫腹神经索(99.36±0.95)、后肠(17.79±3.11)和表皮(9.21±0.12)中的相对表达量较高；在幼虫期随幼虫生长发育表达量逐渐升高，在成虫期的表达量最高。通过喂食2龄幼虫Ldtan的dsRNA能有效降低靶标基因的表达量，使幼虫体色变深呈一定的棕褐色，并且具有一定的致死效应。通过RNAi技术干涉保幼激素合成和信号途径相关基因，发现Ldtan表达量降低，而干扰蜕皮激素合成和信号途径相关基因，Ldtan表达量增加。【结论】结果提示Ldtan参与了马铃薯甲虫的黑色素合成，并且蜕皮激素和保幼激素可能影响其表达。

关键词: 马铃薯甲虫, RNA干扰, NBAD水解酶, 保幼激素, 蜕皮激素

Abstract: 【Aim】 This study aims to clarify the function of N-β-alanyl-dopamine (NBAD) hydrolase gene important in melanin biosythesis in the Colorado potato beetle, Leptinotarsa decemlineata by RNA interference. 【Methods】 The NBAD hydrolase gene in L. decemlineata was characterized by data mining based on its transcriptome, its cDNA was cloned by RT-PCR, and the gene completeness and phylogeny were determined by multiple alignment and phylogenetic analysis, respectively. The expression levels of NBAD hydrolase gene in different developmental stages, tissues of the 4th instar larvae and male gonad and ovary of adults of L. decemlineata were detected by qPCR. The color change during larval growth was observed after RNAi, and the mechanism how the expression of NBAD hydrolase gene was influenced by juvenile hormone (JH) and molting hormone (MH) was assayed .【Results】 An NBAD hydroxylase gene was cloned from L. decemlineata and named Ldtan (GenBank accession no.: KY221866). Its encoded protein shows the highest amino acid sequence identity with the homologous proteins from Tribolium castaneum and Dendroctonus ponderosae and clustered into the same clade with them. The spatial expression profiles showed that Ldtan were highly expressed in ventral nerve cord, hindgut and cuticle of L. decemlineata, with the relative expression levels of 99.36±0.95, 17.79±3.11 and 9.21±0.12, respectively, while the temporal expression profiles showed that its expression level increased along with larval growth and reached the peak at the adult stage. Knockdown of Ldtan gene by feeding dsLdtan to the 2nd instar larvae not only led to tanned color, but also a degree of lethal effect. Knocking down the expression of JH synthesis and signal-related genes by RNAi downregulated the expression of Ldtan, while knocking down the expression of MH synthesis and signal-related genes by RNAi upregulated the expression of Ldtan. 【Conclusion】 The results suggest that Ldtan is involved in melanin synthesis in L. decemlineata, and JH and MH probably regulate its expression.

Key words: Leptinotarsa decemlineata; RNA interference, NBAD hydrolase, juvenile hormone, molting hormone

杜晓燕, 付开赟, 徐晴玉, 吐尔逊·阿合买提, 丁新华, 何江, 郭文超. 马铃薯甲虫N-β-丙酰多巴胺水解酶基因的克隆及功能分析[J]. , 2018, 61(6): 637-645.

DU Xiao-Yan, FU Kai-Bin, XU Qing-Yu, Tursun AHMT, DING Xin-Hua, HE Jiang, GUO Wen-Chao. Molecular cloning and functional characterization of N-β-alanyl-dopamine hydroxylase gene in the Colorado potato beetle, Leptinotarsa decemlineata (Coleoptera: Chrysomelidae)[J]. , 2018, 61(6): 637-645.

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http://www.insect.org.cn/CN/Y2018/V61/I6/637

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马铃薯甲虫N-β-丙酰多巴胺水解酶基因的克隆及功能分析

Molecular cloning and functional characterization of N-β-alanyl-dopamine hydroxylase gene in the Colorado potato beetle, Leptinotarsa decemlineata (Coleoptera: Chrysomelidae)

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