褐飞虱一个新的海藻糖合成酶基因的特性、 发育表达及RNAi效果分析

doi:10.3864/j.issn.0578-1752.2019.03.007

摘要/Abstract

摘要：

背景昆虫海藻糖合成酶基因是昆虫海藻糖合成的主要基因,大多数昆虫中只拥有一个海藻糖-6-磷酸合成酶（trehalose-6-phosphate synthase,TPS）基因,部分昆虫存在一个海藻糖-6-磷酸酯酶（trehalose-6-phosphate phosphatase,TPP）基因。前期研究发现褐飞虱（Nilaparvata lugens）中拥有两个TPS,对其功能研究发现TPS不仅能够调控海藻糖代谢,还可介导海藻糖酶调控几丁质合成与降解途径,控制昆虫的蜕皮过程。目的通过对褐飞虱转录组测序分析获得了一个新的TPS,检测该基因在褐飞虱不同发育阶段的表达情况,探究该基因的功能与前期发现的两个TPS的区别。方法对获得的新TPS基因序列采用克隆技术获得全长cDNA序列,经验证正确后,对其蛋白的一级、二级、三级结构及与其他昆虫的TPS进行比对分析,最后采用实时荧光定量PCR（qRT-PCR）技术测定3个不同TPS在褐飞虱不同发育阶段的表达,并采用RNA干扰（RNAi）技术抑制TPS3的表达。结果在前期研究的基础上,克隆出一个新的TPS,并命名为TPS3。TPS3开放阅读框长度为2 352 bp,编码783个氨基酸,预测蛋白分子量为88.9 kD,等电点为5.47,具有亲水性结构。生物信息学分析表明,褐飞虱3个TPS蛋白具有较高的同源性,都具有TPS和TPP两个保守结构域及其他特征序列,并且α-螺旋、β-折叠和无规则卷曲所占的比例较为接近。褐飞虱不同发育阶段3条TPS的相对表达量不同,TPS1的相对表达量从4龄0 h开始逐渐上升,至成虫阶段达到最高,TPS2的相对表达量从4龄末期开始明显上升且在整个5龄阶段都有较高的表达,TPS3的相对表达量在5龄末期和成虫初期较高。单独干扰褐飞虱TPS3 48 h后被干扰基因的相对表达量下降,dsTPS3能有效抑制TPS3的表达。结论在褐飞虱中发现一个新的TPS（TPS3）,其与褐飞虱中已经报道的TPS1和TPS2具有较高的同源性。不同发育阶段表达结果表明,3个TPS在发育过程中行使的功能不同。RNAi能够有效抑制TPS3的表达并导致褐飞虱蜕皮障碍和翅发育畸形。

关键词: 褐飞虱, 海藻糖-6-磷酸合成酶, 序列分析, 蛋白结构, 实时荧光定量PCR

Abstract:

【Background】 Insect trehalose-6-phosphate synthase (TPS) is the main gene for insect trehalose synthesis. Most insects possess one trehalose-6-phosphate synthase gene, and some insects have one trehalose-6-phosphate phosphatase (TPP) gene. Previous studies have found that there are two TPS genes in the brown planthopper (Nilaparvata lugens) and their functional studies have shown that TPS can not only regulate trehalose metabolism, but also mediate trehalase regulation of chitin synthesis and degradation pathways, and control insect molting process.【Objective】 A novel TPS was obtained by the transcriptome sequencing of N. lugens. The objective of this study is to detect the expression of the gene in different developmental stages of N. lugens, and to explore the difference between the function of the gene and the two previously discovered TPS genes.【Method】 The full-length cDNA sequence of the novel TPS was obtained by cloning technique. After correct verification, the primary, secondary, and tertiary structures of the protein were obtained. The protein structure and alignment of TPS were analyzed with other insects. Finally, qRT-PCR was used to determine the expression of three different TPS genes at different development stages of N. lugens, and RNA interference (RNAi) was used to inhibit the expression of TPS3.【Result】 Based on the previous study, this novel TPS was cloned and named as TPS3. The open reading frame of TPS3 is 2 352 bp, encoding 783 amino acids. The predicted molecular weight of the protein is 88.9 kD and the isoelectric point is 5.47, with a hydrophilic structure. Bioinformatics analysis showed that the three TPS proteins of N. lugens had high homology, with two conserved domains of TPS and TPP and other characteristic sequences, and the proportion of α-helix, β-sheet, and random curl was similar. The relative expression levels of three TPS genes in different developmental stages of N. lugens were different. The relative expression level of TPS1 gradually increased from 0 h of 4th instar to the highest level at the adult stage. The relative expression level of TPS2 increased obviously from the end of 4th instar and was higher in the whole 5th instar. The expression of TPS3 was relatively higher at the end of the 5th instar and the early stage of adult. The relative expression level of the interfered gene significantly decreased after 48 hours of single interference with TPS3. The dsTPS3 could effectively inhibit the expression of TPS3.【Conclusion】A novel TPS (TPS3) was found in N. lugens, which has high homology with TPS1 and TPS2 reported in N. lugens. The results of expression at different developmental stages showed that the three TPS genes performed different functions during development. RNAi could effectively inhibit the expression of TPS3 and lead to molting and wing deformity.

Key words: Nilaparvata lugens, trehalose-6-phosphate synthase (TPS), sequence analysis, protein structure, quantitative real-time PCR (qRT-PCR)

唐斌,沈祺达,曾伯平,肖仲久,邱玲玉,潘碧莹,李昆,张道伟. 褐飞虱一个新的海藻糖合成酶基因的特性、发育表达及RNAi效果分析[J]. 中国农业科学, 2019, 52(3): 466-477.

TANG Bin,SHEN QiDa,ZENG BoPing,XIAO ZhongJiu,QIU LingYu,PAN BiYing,LI Kun,ZHANG DaoWei. Characteristics, Developmental Expression and RNAi Effect Analysis of a Novel Trehalose-6-Phosphate Synthase Gene in Nilaparvata lugens[J]. Scientia Agricultura Sinica, 2019, 52(3): 466-477.

图/表 9

表1

图1

图2

表2

图3

表3

图4

图5

图6

参考文献 48

[1]	BECKER A ,SCHLÖDER P,STEELE J E,WEGENER G. The regulation of trehalose metabolism in insects. Experientia, 1996,52(5):433-439.
[2]	ELBEIN A D, PAN Y T, PASTUSZAK I, CARROLL D . New insights on trehalose: a multifunctional molecule. Glycobiology, 2003,13(4):17R-27R. doi: 10.1093/glycob/cwg047 pmid: 12626396
[3]	TANG B, CHEN J, YAO Q, PAN Z Q, XU W H, WANG S G, ZHANG W Q . Characterization of a trehalose-6-phosphate synthase gene from Spodoptera exigua and its function identification through RNA interference. Journal of Insect Physiology, 2010,56(7):813-821.
[4]	张露, 朱世城, 郑好, 沈祺达, 王世贵, 唐斌 . 褐飞虱海藻糖酶基因在表皮几丁质代谢中的调控作用. 中国农业科学, 2017,50(6):1047-1056.
	ZHANG L, ZHU S C, ZHENG H, SHEN Q D, WANG S G, TANG B . Regulatory function of trehalase genes on chitin metabolism in the cuticle of Nilaparvata lugens. Scientia Agricultura Sinica, 2017,50(6):1047-1056. (in Chinese)
[5]	STEELE J E . Occurrence of a hyperglycemic factor in the corpus cardiacum of an insect. Nature, 1961,192:680-681. doi: 10.1038/192680a0
[6]	BOWERS W S, FRIEDMAN S . Mobilization of fat body glycogen by an extract of corpus cardiacum. Nature, 1963,198:685. doi: 10.1038/198685a0
[7]	陈静 . 褐飞虱遗传多样性及其海藻糖合成酶基因功能的分析[D]. 广州: 中山大学, 2010.
	CHEN J . Genetic diversity and function analysis of a trehalose phosphate synthase gene of Nilaparvata lugens[D]. Guangzhou: Sun Yat-Sen University, 2010. ( in Chinese)
[8]	唐斌, 徐青叶, 赵丽娜, 王世贵, 张帆 . 昆虫海藻糖及其合成酶基因的特性与功能研究进展. 应用昆虫学报, 2014,51(6):1397-1405.
	TANG B, XU Q Y, ZHAO L N, WANG S G, ZHANG F . Progress in research on the characteristics and functions of trehalose and the TPS gene in insects. Chinese Journal of Applied Entomology, 2014,51(6):1397-1405. (in Chinese)
[9]	THOMPSON S N . Trehalose - The insect ‘blood’ sugar. Advances in Insect Physiology, 2003,31:205-285.
[10]	唐斌, 张露, 熊旭萍, 汪慧娟, 王世贵 . 海藻糖代谢及其调控昆虫几丁质合成研究进展. 中国农业科学, 2018,51(4):697-707.
	TANG B, ZHANG L, XIONG X P, WANG H J, WANG S J . Advances in trehalose metabolism and its regulation of insect chitin synthesis. Scientia Agricultura Sinica, 2018,51(4):697-707. (in Chinese)
[11]	于彩虹, 卢丹, 林荣华, 王晓军, 姜辉, 赵飞 . 海藻糖——昆虫的血糖. 昆虫知识, 2008,45(5):832-837.
	YU C H, LU D, LIN R H, WANG X J, JIANG H, ZHAO F . Trehalose——The blood sugar in insects. Chinese Bulletin of Entomology, 2008,45(5):832-837. (in Chinese)
[12]	张道伟, 陈静, 郭玉双 . 白背飞虱海藻糖合成酶基因的克隆及序列分析. 黑龙江农业科学, 2012(5):14-19.
	ZHANG D W, CHEN J, GUO Y S . Cloning and sequence analysis oftrehalose phosphate synthase gene from Sogatella furcifera. Heilongjiang Agricultural Sciences, 2012(5):14-19. (in Chinese)
[13]	赵丽娜 . 海藻糖酶和海藻糖合成酶基因对褐飞虱海藻糖代谢途径关键基因的调控研究[D]. 杭州: 杭州师范大学, 2014.
	ZHAO L N . Regulating effects of trehalase and TPS on the key genes in the pathway of trehalose metabolic in Nilaparvata lugens[D]. Hangzhou: Hangzhou Normal University, 2014. ( in Chinese)
[14]	陈静, 张道伟 . 德国小蠊两个海藻糖合成酶基因的克隆、组织分布及温度诱导表达分析. 昆虫学报, 2015,58(10):1046-1053.
	CHEN J, ZHANG D W . Molecular cloning, tissue distribution and temperature-induced expression of two trehalose-6-phosphate synthase genes in Blattella germanica(Blattodea: Blattellidae). Acta Entomologica Sinica, 2015,58(10):1046-1053. (in Chinese)
[15]	XIONG K C, WANG J, LI J H, DENG Y Q, PU P, FAN H, LIU Y H. RNA interference of a trehalose-6-phosphate synthase gene reveals its role during larval-pupal metamorphosis in Bactrocera minax(Diptera:Tephritidae) . Journal of Insect Physiology, 2016,91/92:84-92.
[16]	SHI J F, XU Q Y, SUN Q K, MENG Q W, MU L L, GUO W C, LI G Q . Physiological roles of trehalose in Leptinotarsa larvae revealed by RNA interference of trehalose-6-phosphate synthase and trehalase genes. Insect Biochemistry and Molecular Biology, 2016,77:52-68. doi: 10.1016/j.ibmb.2016.07.012 pmid: 27524277
[17]	YANG M M, ZHAO L N, SHEN Q D, XIE G Q, WANG S G, TANG B . Knockdown of two trehalose-6-phosphate synthases severely affects chitin metabolism gene expression in the brown planthopper Nilaparvata lugens. Pest Management Science, 2017,73(1):206-216. doi: 10.1002/ps.4287 pmid: 27060284
[18]	CHEN Q W, JIN S, ZHANG L, SHEN Q D, WEI P, WEI C M, WANG S G, TANG B . Regulatory functions of trehalose-6-phosphate synthase in the chitin biosynthesis pathway in Tribolium castaneum(Coleoptera: Tenebrionidae) revealed by RNA interference. Bulletin of Entomological Research, 2018,108(3):388-399. doi: 10.1017/S000748531700089X pmid: 28920565
[19]	余德涛 . 褐飞虱的生物学特征及鉴别. 农技服务, 2007,24(2):45-46.
	YU D T . Biological characteristics and identification of brown planthopper. Agricultural Technology Service, 2007,24(2):45-46. (in Chinese)
[20]	高希武, 彭丽年, 梁帝允 . 对2005年水稻褐飞虱大发生的思考. 植物保护, 2006,32(2):23-24.
	GAO X W, PENG L N, LIANG D Y . Factors causing the outbreak of brown planthopper (BHP), Nilapavata lugens Stå1 in China in 2005. Plant Protection, 2006,32(2):23-24. (in Chinese)
[21]	LIU Z W, HAN Z J . Fitness costs of laboratory-selected imidacloprid resistance in the brown planthopper, Nilaparvata lugens Stål. Pest Management Science, 2006,62(3):279-282.
[22]	齐国君 . 褐飞虱发生规律的再研究——广西、安徽两省区的个例分析[D]. 南京: 南京农业大学, 2009.
	QI G J . New study on population dynamics of brown planthopper Nilaparvata lugens-A case study of Guangxi and Anhui[D]. Nanjing: Nanjing Agricultural University, 2009. ( in Chinese)
[23]	范峰峰, 邱颖波, 刘行丹, 李绍清, 刘建丰 . 水稻抗褐飞虱基因及其育种应用研究进展. 中国农学通报, 2014,30(6):13-19.
	FAN F F, QIU Y B, LIU X D, LI S Q, LIU J F . Research progress on the research and use of brown planthopper resistance genes in rice. Chinese Agricultural Science Bulletin, 2014,30(6):13-19. (in Chinese)
[24]	俞姗姗, 刘雅, 杨萌萌, 沈祺达, 谢国强, 王世贵, 唐斌 . 褐飞虱在不同水稻品系上繁殖能力及相关解毒酶系基因表达变化分析. 杭州师范大学学报(自然科学版), 2016,15(6):595-599.
	YU S S, LIU Y, YANG M M, SHEN Q D, XIE G Q, WANG S G, TANG B . Fertility and related detoxification enzyme systems gene expression of Nilaparvata lugens feeding on different rice varieties. Journal of Hangzhou Normal University (Natural Science Edition), 2016,15(6):595-599. (in Chinese)
[25]	程遐年, 吴进才, 马飞 . 褐飞虱研究与防治. 北京: 中国农业出版社, 2003.
	CHENG X N, WU J C, MA F. Brown Planthopper:Occurrence and Control. Beijing:China Agriculture Press, 2003. ( in Chinese)
[26]	李汝铎, 丁锦华, 胡国文, 苏德明 . 褐飞虱及其种群管理. 上海: 复旦大学出版社, 1996.
	LI R D, DING J H, HU G W, SU D M. The Brown Planthopper and Its Population Management. Shanghai: Fudan University Press, 1996. ( in Chinese)
[27]	杨萌萌 . 海藻糖酶及其抑制剂 (Validamycin)对褐飞虱海藻糖和几丁质代谢的调控研究[D]. 杭州: 杭州师范大学, 2016.
	YANG M M . Regulating effects of trehalase and its inhibitor (Validamycin) on the trehalose and chitin metabolism in Nilaparvata lugens[D]. Hangzhou: Hangzhou Normal University, 2016. ( in Chinese)
[28]	XUE J, ZHOU X, ZHANG CX, YU L L, FAN H W, WANG Z, XU H J, XI Y, ZHU Z R, ZHOU W W, PAN P L, LI B L, COLBOURNE J K, NODA H, SUETSUGU Y, KOBAYASHI T, ZHENG Y, LIU S, ZHANG R, LIU Y, LUO Y D, FANG D M, CHEN Y, ZHAN D L, LV X D, CAI Y, WANG Z B, HUANG H J, CHENG R L, ZHANG X C, LOU Y H, YU B, ZHOU J C, YE Y X, ZHANG W Q, SHEN Z C, YANG H M, WANG J, WANG J, BAO Y Y, CHENG J A . Genomes of the rice pest brown planthopper and its endosymbionts reveal complex complementary contributions for host adaptation. Genome Biology, 2014,15(12):521. doi: 10.1186/s13059-014-0521-0 pmid: 4269174
[29]	XU H J, XUE J, LU B, ZHANG X C, ZHOU J C, HE S F, MA X F, JIANG Y Q, FAN H W, XU J Y, YE Y X, PAN PL, LI Q, BAO Y Y, NIJHOUT H F, ZHANG C X . Two insulin receptors determine alternative wing morphs in planthoppers. Nature, 2015,519(7544):464-467. doi: 10.1038/nature14286 pmid: 25799997
[30]	LIN X D, XU Y L, JIANG J R, LAVINE M, LAVINE L C . Host quality induces phenotypic plasticity in a wing polyphonic insect. Proceedings of the National Academy of Sciences of the United States of America, 2018,115(29):7563-7568. doi: 10.1073/pnas.1721473115
[31]	TANG B, WANG S, WANG S G, WANG H J, ZHANG J Y, CUI S Y . Invertebrate trehalose-6-phosphate synthase gene: genetic architecture, biochemistry, physiological function, and potential applications. Frontiers in Physiology, 2018,9:30. doi: 10.3389/fphys.2018.00030 pmid: 5797772
[32]	ARNOLD K, BORDOLI L, KOPP J, SCHWEDE T . The SWISS-MODEL workspace: a web-based environment for protein structure homology modelling. Bioinformatics, 2006,22(2):195-201. doi: 10.1093/bioinformatics/bti770 pmid: 16301204
[33]	RAO K N, KUMARAN D, SEETHARAMAN J, BONANNO J B, BURLEY S K, SWAMINATHAN S . Crystal structure of trehalose-6- phosphate phosphatase-related protein: Biochemical and biological implications. Protein Science, 2006,15(7):1735-1744.
[34]	BENKERT P, BIASINI M, SCHWEDE T . Toward the estimation of the absolute quality of individual protein structure models. Bioinformatics, 2011,27(3):343-350. doi: 10.1093/bioinformatics/btq662 pmid: 21134891
[35]	BIASINI M, BIENERT S, WATERHOUSE A, ARNOLD K, STUDER G, SCHMIDT T, KIEFER F, CASSARINO T G, BERTONI M, BORDOLI L, SCHWEDE T . SWISS-MODEL: modelling protein tertiary and quaternary structure using evolutionary information. Nucleic Acids Research, 2014,42(W1):252-258. doi: 10.1093/nar/gku340 pmid: 24782522
[36]	ZHAO L N, YANG M M, SHEN Q D, LIU X, SHI Z K, WANG S G, TANG B . Functional characterization of three trehalase genes regulating the chitin metabolism pathway in rice brown planthopper using RNA interference. Science Reports, 2016,6:27841. doi: 10.1038/srep27841 pmid: 27328657
[37]	TANG B, ZHENG H Z, XU Q, ZOU Q, WANG G J, ZHANG F, WANG S G, ZHANG Z H . Cloning and pattern of expression of trehalose-6-phosphate synthase cDNA from Catantops pinguis(Orthoptera: Catantopidae). European Journal of Entomology, 2011,108(3):355-363.
[38]	崔淑燕 . 东亚飞蝗海藻糖合成酶基因的克隆与功能研究[D]. 重庆: 重庆大学, 2008.
	CUI S Y . Isolation, cloning and function analysis of trehalose-6- phosphate synthase from Locusta migratoria manilensis[D]. Chongqing: Chongqing University, 2008. ( in Chinese)
[39]	KERN C, WOLF C, BENDER F, BERGER M, NOACK S, SCHMALZ S, ILG T . Trehalose-6-phosphate synthase from the cat flea Ctenocephalides felis and Drosophila melanogaster: gene identification, cloning, heterologous functional expression and identification of inhibitors by high throughput screening. Insect Molecular Biology, 2012,21(4):456-471.
[40]	李源, 郝友进, 张玉娟, 司风玲, 陈斌 . 葱蝇海藻糖-6-磷酸合成酶基因的克隆、序列分析及滞育相关表达. 昆虫学报, 2013,56(4):329-338.
	LI Y, HAO Y J, ZHANG Y J, SI F L, CHEN B . Cloning, bioinformatics analysis and diapause-related expression of trehalose- 6-phosphate synthase gene from the onion maggot,Delia antiqua(Diptera: Anthomyiidae). Acta Entomologica Sinica, 2013,56(4):329-338. (in Chinese)
[41]	GUO Q, HAO Y J, LI Y, ZHANG Y J, REN S, SI F L, CHEN B . Gene cloning, characterization and expression and enzymatic activities related to trehalose metabolism during diapause of the onion maggot Delia antiqua(Diptera: Anthomyiidae). Gene, 2015,565(1):106-115. doi: 10.1016/j.gene.2015.03.070 pmid: 25841989
[42]	SHI Q, CHUNG J S . Trehalose metabolism in the blue crab Callinectes sapidus: isolation of multiple structural cDNA isoforms of trehalose-6-phosphate synthase and their expression in muscles. Gene, 2014,536(1):105-113.
[43]	DMITRYJUK M, ŁOPIENSKA-BIERNAT E, ZAOBIDNA E A . The in vitro effect of ivermectin on the activity of trehaose synthesis pathway enzymes and their mRNA expression in the muscle of adult female Ascaris suum (Nematoda) . The Scientific World Journal, 2014, 2014: Article ID 936560. doi: 10.1155/2014/936560 pmid: 4227463
[44]	XU J, BAO B, ZHANG Z F, YI Y Z, XU W H . Identification of a novel gene encoding the trehalose phosphate synthase in the cotton bollworm, Helicoverpa armigera. Glycobiology, 2009,19(3):250-257. doi: 10.1093/glycob/cwn127 pmid: 19004876
[45]	YOSHIDA M, MATSUDA H, KUBO H, NISHIMURA T . Molecular characterization of Tps1 and Treh genes in Drosophila and their role in body water homeostasis. Science Reports, 2016,6:30582. doi: 10.1038/srep30582 pmid: 27469628
[46]	CHUNG J S . A trehalose6-phosphate synthase gene of the hemocytes of the blue crab,Callinectes sapidus: cloning, the expression, its enzyme activity and relationship to hemolymph trehalose levels. Saline Systems, 2008,4:18. doi: 10.1186/1746-1448-4-18 pmid: 19077285
[47]	MATSUDA H, YAMADA T, YOSHIDA M, NISHIMURA T . Flies without trehalose. The Journal of Biological Chemistry, 2015,290(2):1244-1255.
[48]	ZHANG L, QIU L Y, YANG H L, WANG H J, ZHOU M, WANG S G, TANG B . Study on the effect of wing bud chitin metabolism and its developmental network genes in the brown planthopper,Nilaparvata lugens by knockdown TRE gene. Frontiers in Physiology, 2017,8:750. doi: 10.3389/fphys.2017.00750 pmid: 5627005

基因名称 Primer name	正向引物 Forward primer (5′-3′)	反向引物 Reverse primer (5′-3′)
QNl18S QNlTPS1 QNlTPS2 QNlTPS3	CGCTACTACCGATTGAA AAGACTGAGGCGAATGGT AGAGTGGACCGCAACAACA GTGATGCGTCGGTGGCTAT	GGAAACCTTGTTACGACTT AAGGTGGAAATGGAATGTG TCAACGCCGAGAATGACTT CCGTTCATCATTGGGCATAGT
dsNlTPS3	GAGTCTGACCTGATAGCCTTTA	ATCGGAGTCCATTTAGTTGT
dsNlTPS3-T7	GGATCCTAATACGACTCACTATAGGGAGTCTGACCTGATAGCCTTTA	GGATCCTAATACGACTCACTATAGGATCGGAGTCCATTTAGTTGT
dsNlGFP	AAGGGCGAGGAGCTGTTCACCG	CAGCAGGACCATGTGATCGCGC
dsNlGFP-T7	GGATCCTAATACGACTCACTATAGGAAGGGCGAGGAGCTGTTCACCG	GGATCCTAATACGACTCACTATAGGCAGCAGGACCATGTGATCGCGC

蛋白名称 Protein name	氨基酸数 Amino acid number	相对分子量 Relative molecular weight	等电点 Isoelectric point	亲水性 Hydrophily
TPS1	807	90536	6.14	-0.180
TPS2	820	93215	6.07	-0.190
TPS3	783	88909	5.47	-0.126

	α-螺旋 α-helix (%)	β-折叠 β-sheet (%)	无规则卷曲 Random coil (%)
TPS1	40.27	12.76	45.35
TPS2	36.59	13.29	49.39
TPS3	35.50	18.01	46.49