两个褐飞虱海藻糖转运蛋白基因的结构及调控海藻糖代谢功能

doi:10.3864/j.issn.0578-1752.2020.23.007

摘要/Abstract

摘要：

【目的】海藻糖是昆虫中的主要血糖物质,在昆虫的发育及生理活动中发挥重要功能。其中,海藻糖转运蛋白（Tret）在将海藻糖从其生成组织（例如脂肪体）运输到其消耗组织的过程中起着重要作用。本研究通过分析褐飞虱（Nilaparvata lugens）两条Tret1的序列结构,进一步抑制NlTret1的表达,探讨这两个NlTret1在褐飞虱体内的生物学功能。【方法】以褐飞虱两条Tret1序列为研究对象,利用生物信息学技术分析其蛋白结构以及与其他昆虫之间的同源性。采用RNAi（RNA interference）技术将合成的外源dsRNA（double-stranded RNA）注射到实验室饲养褐飞虱种群体内,抑制其体内NlTret1的表达,分别在注射48 h后取材,抽取总RNA并用反转录试剂盒合成第一链cDNA,采用qRT-PCR（quantitative real-time PCR）技术检测dsNlTret1的干扰效果以及RNAi后褐飞虱体内海藻糖代谢通路中相关基因的表达,最后测定葡萄糖、海藻糖和糖原含量以及海藻糖酶活性。【结果】生物信息学分析表明,NlTret1-like X1和NlTret1-2 X1的开放阅读框长度分别为1 920和1 578 bp,分别编码639和525个氨基酸,预测蛋白分子量分别为69.29和58.71 kD,等电点分别为8.32和8.36;NlTret1-like X1和NlTret1-2 X1的二级结构主要包含螺旋和卷曲;保守结构域分析显示它们均属于MFS家族。进化分析结果显示,不同昆虫的Tret1蛋白具有较高的同源性,且褐飞虱与其他半翅目昆虫具有较近的亲缘关系;与注射dsGFP组相比,注射靶标基因的dsRNA后均能够显著沉默本基因的表达;褐飞虱体内糖原、葡萄糖在注射dsNlTret1-like X1和dsNlTret1-2 X1后,其含量均无显著变化,然而不同于注射dsNlTret1-2 X1组,在注射dsNlTret1-like X1后褐飞虱体内海藻糖含量极显著升高;干扰NlTret1-like X1 48 h后褐飞虱体内TPS1、TPS2、TRE1-1、TRE1-2和TRE2的表达均极显著下调;而干扰NlTret1-2 X1 48 h后褐飞虱体内TPS1、TPS2和TRE1-1的表达虽也极显著下降,但TRE1-2和TRE2极显著上调;在注射dsNlTret1-like X1后,试虫可溶性海藻糖酶和膜结合型海藻糖酶的活性均极显著降低,而在注射dsNlTret1-2 X1后无显著变化。【结论】褐飞虱的两个Tret1在不同组织间发挥着不同的功能,其中NlTret1-like X1在特异性转运海藻糖参与能量供应中起到更为显著的作用。研究结果有助于探索Tret1在昆虫或无脊椎动物中调控海藻糖代谢平衡的调节机制,可为将来通过调控血糖平衡来控制褐飞虱等害虫提供理论依据。

关键词: 褐飞虱, 海藻糖转运蛋白, 结构, 海藻糖代谢, RNA干扰

Abstract:

【Objective】 Trehalose is the main blood sugar substance in insects and plays an important role in insect development and physiological activities. Among them, trehalose transporter (Tret) plays a key role in the transportation of trehalose from trehalose-producing tissues (such as fat body) to trehalose-consuming tissues. The objective of this study is to explore the biological functions of these two NlTret1s in brown planthopper (Nilaparvata lugens) by analyzing the sequence structure of two NlTret1s and further suppressing the expression of NlTret1s. 【Method】Taking these two NlTret1 sequences as the research object, the protein structure and homology with other insects were analyzed by bioinformatics technology. The RNA interference (RNAi) technology was used to inject the synthetic exogenous dsRNA (double-stranded RNA) into the laboratory feeding population of N. lugens, to inhibit the expression of the NlTret1s. The total RNA was extracted to synthesize the first-strand cDNA using reverse transcription Kit, qRT-PCR (quantitative real-time PCR) technology was used to detect the RNAi effect of dsNlTret1s and the expression of related genes in the trehalose metabolism pathway in N. lugens after RNAi, and finally glucose, trehalose and glycogen content as well as trehalase enzyme activity were determined. 【Result】 Bioinformatics analysis showed that the open reading frames of NlTret1-like X1 and NlTret1-2 X1 are 1 920 and 1 578 bp in length, encoding 639 and 525 amino acids, respectively. The predicted protein molecular weights are 69.29 and 58.71 kD, and the isoelectric points are 8.32 and 8.36, respectively. The secondary structure of NLTret1-like X1 and NLTret1-2 X1 is mainly composed of helix and coil. Conservative domain analysis showed that they all belong to the MFS family. The results of evolutionary analysis showed that Tret1 of different insects had high homology, and N. lugens was closely related to other hemiptera insects. Compared with the dsGFP group, the target gene was inhibited significantly after injection with dsNlTret1-like X1 or dsNlTret1-2 X1. Furthermore, the content of glycogen and glucose in N. lugens did not change significantly, but unlike the dsNlTret1-2 X1 group, the trehalose content of N. lugens was significantly increased with the injection of dsNlTret1-like X1. Meanwhile, the expression of the TPS1, TPS2, TRE1-1, TRE1-2 and TRE2 in N. lugens was significantly down-regulated after the NlTret1-like X1 knocked down for 48 h. The expression of TPS1, TPS2 and TRE1-1 in N. lugens also decreased significantly after injection with dsNlTret1-2 X1 for 48 h, while TRE1-2 and TRE2 showed a very significant upward trend. Moreover, after injection of dsNlTret1-like X1, the activities of soluble trehalase and membrane-bound trehalase were significantly reduced, but there was no significant change after injection with dsNlTret1-2 X1. 【Conclusion】These two Tret1s of N. lugens play different functions in different tissues, among which NlTret1-like X1 plays a more significant role in the specific transport of trehalose involved in energy supply. The results are helpful to explore the regulatory mechanism of Tret1 regulating the balance of trehalose metabolism in insects or invertebrates, and provide a theoretical basis for the future control of pests by regulating blood sugar balance, such as N. lugens.

Key words: brown planthopper (Nilaparvata lugens), trehalose transporter (Tret), structure, trehalose metabolism, RNAi

於卫东,潘碧莹,邱玲玉,黄镇,周泰,叶林,唐斌,王世贵. 两个褐飞虱海藻糖转运蛋白基因的结构及调控海藻糖代谢功能[J]. 中国农业科学, 2020, 53(23): 4802-4812.

YU WeiDong,PAN BiYing,QIU LingYu,HUANG Zhen,ZHOU Tai,YE Lin,TANG Bin,WANG ShiGui. The Structure Characteristics and Biological Functions on Regulating Trehalose Metabolism of Two NlTret1s in Nilaparvata lugens[J]. Scientia Agricultura Sinica, 2020, 53(23): 4802-4812.

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参考文献 37

[1]	徐春春, 纪龙, 陈中督, 方福平 . 2017年我国水稻产业形势分析及2018年展望. 中国稻米, 2018,24(2):1-3.
	XU C C, JI L, CHEN Z D, FANG F P . Analysis of China’s rice industry in 2017 and the outlook for 2018. China Rice, 2018,24(2):1-3. (in Chinese)
[2]	CHENG X Y, ZHU L L, HE G C . Towards understanding of molecular interactions between rice and the brown planthopper. Molecular Plant, 2013,6(3):621-634. doi: 10.1093/mp/sst030
[3]	LU G, ZHANG T, HE Y, ZHOU G . Virus altered rice attractiveness to planthoppers is mediated by volatiles and related to virus titre and expression of defense and volatile-biosynthesis genes. Scientific Reports, 2016,6:38581. doi: 10.1038/srep38581 pmid: 27924841
[4]	BODDUPALLY D, TAMIRISA S, GUNDRA S R, VUDEM D R, KHAREEDU V R . Expression of hybrid fusion protein (Cry1Ac::ASAL) in transgenic rice plants imparts resistance against multiple insect pests. Scientific Reports, 2018,8:8458. doi: 10.1038/s41598-018-26881-9 pmid: 29855556
[5]	吴碧球, 黄所生, 黄凤宽 . 环境因素对水稻品种抗褐飞虱的影响研究概况. 植物保护, 2015,41(1):1-6.
	WU B Q, HUANG S S, HUANG F K . A review on factors affecting resistance of rice varieties to the rice brown planthopper. Plant Protection, 2015,41(1):1-6. (in Chinese)
[6]	TANAKA K, ENDO S, KAZANO H . Toxicity of insecticides to predators of rice planthoppers: Spiders, the mirid bug and the dryinid wasp. Applied Entomology and Zoology, 2000,35(1):177-187. doi: 10.1303/aez.2000.177
[7]	ROLA A C, PINGALI P L . Pesticides, Rice Productivity, and Farmers’ Health: An Economic Assessment. Manila, Philippines: International Rice Research Institute, 1993.
[8]	NAUEN R, DENHOLM I . Resistance of insect pests to neonicotinoid insecticides: Current status and future prospects. Archives of Insect Biochemistry and Physiology, 2005,58(4):200-215. doi: 10.1002/arch.20043 pmid: 15756698
[9]	WANG H Y, YANG Y, SU J Y, SHEN J L, GAO C F, ZHU Y C . Assessment of the impact of insecticides on Anagrus nilaparvatae (Pang et Wang) (Hymenoptera: Mymanidae), an egg parasitoid of the rice planthopper, Nilaparvata lugens (Hemiptera: Delphacidae). Crop Protection, 2008,27(3/5):514-522. doi: 10.1016/j.cropro.2007.08.004
[10]	BOTTRELL D G, SCHOENLY K G . Resurrecting the ghost of green revolutions past: The brown planthopper as a recurring threat to high-yielding rice production in tropical Asia. Journal of Asia-Pacific Entomology, 2012,15(1):122-140. doi: 10.1016/j.aspen.2011.09.004
[11]	BECKER A, SCHLÖDER P, STEELE J E, WEGENER G . The regulation of trehalose metabolism in insects. Experientia, 1996,52(5):433-439. doi: 10.1007/BF01919312 pmid: 8706810
[12]	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
[13]	IORDACHESCU M, IMAI R . Trehalose biosynthesis in response to abiotic stresses. Journal of Integrative Plant Biology, 2008,50(10):1223-1229. doi: 10.1111/j.1744-7909.2008.00736.x
[14]	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. doi: 10.1016/j.jinsphys.2010.02.009 pmid: 20193689
[15]	SHUKLA E, THORAT L J, NATH B B, GAIKWAD S M . Insect trehalase: Physiological significance and potential applications. Glycobiology, 2015,25(4):357-367. doi: 10.1093/glycob/cwu125 pmid: 25429048
[16]	AVONCE N, MENDOZA-VARGAS A, MORETT E, ITURRIAGA G . Insights on the evolution of trehalose biosynthesis. BMC Evolutionary Biology, 2006,6:109. doi: 10.1186/1471-2148-6-109 pmid: 17178000
[17]	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: 29445344
[18]	刘贻聪 . 丽蚜小蜂取食糖分对寄生烟粉虱的影响及糖转运蛋白诱导表达分析[D]. 北京: 中国农业科学院, 2018.
	LIU Y C . Effects of sugar diets on the parasitism of Encarsia formosa on Bemisia tabaci and analysis of induced expression of sugar transporter gene[D]. Beijing: Chinese Academy of Agricultural Sciences, 2018. (in Chinese)
[19]	郭婧, 范宇鸿, 宋立猛 . 葡萄糖转运蛋白-1在肿瘤中的表达及意义. 国际检验医学杂志, 2019,40(16):2009-2011, 2034.
	GUO J, FAN Y H, SONG L M . Expression and significance of glucose transporter-1 in tumors. International Journal of Laboratory Medicine, 2019,40(16):2009-2011, 2034. (in Chinese)
[20]	杨泽众 . Q烟粉虱与内共生细菌互作机制及B烟粉虱糖转运蛋白超家族注释与功能研究[D]. 长沙: 湖南农业大学, 2017.
	YANG Z Z . Symbiotic relationship between Bemisia tabaci B and its endosymbionts and annotation and function research of sugar transporter superfamily of Bemisia tabaci B[D]. Changsha: Hunan Agricultural University, 2017. (in Chinese)
[21]	KIKAWADA T, SAITO A, KANAMORI Y, NAKAHARA Y, IWATA K, TANAKA D, WATANABE M, OKUDA T . Trehalose transporter 1, a facilitated and high-capacity trehalose transporter, allows exogenous trehalose uptake into cells. Proceedings of the National Academy of Sciences of the United States of America, 2007,104(28):11585-11590. doi: 10.1073/pnas.0702538104 pmid: 17606922
[22]	KIKUTA S, NAKAMURA Y, HATTORI M, SATO R, KIKAWADA T, NODA H . Herbivory-induced glucose transporter gene expression in the brown planthopper, Nilaparvata lugens. Insect Biochemistry and Molecular Biology, 2015,64:60-67. doi: 10.1016/j.ibmb.2015.07.015 pmid: 26226652
[23]	PRICE D R, WILKINSON H S, GATEHOUSE J A . Functional expression and characterisation of a gut facilitative glucose transporter, NlHT1, from the phloem-feeding insect Nilaparvata lugens (rice brown planthopper). Insect Biochemistry and Molecular Biology, 2007,37(11):1138-1148. doi: 10.1016/j.ibmb.2007.07.001
[24]	KIKUTA S, KIKAWADA T, HAGIWARA-KOMODA Y, NAKASHIMA N, NODA H . Sugar transporter genes of the brown planthopper, Nilaparvata lugens: A facilitated glucose/fructose transporter. Insect Biochemistry and Molecular Biology, 2010,40(11):805-813. doi: 10.1016/j.ibmb.2010.07.008
[25]	PRICE D R, KARLEY A J, ASHFORD D A, ISAACS H V, POWNALL M E, WILKINSON H S, GATEHOUSE J A, DOUGLAS A E . Molecular characterisation of a candidate gut sucrase in the pea aphid, Acyrthosiphon pisum. Insect Biochemistry and Molecular Biology, 2007,37(4):307-317. doi: 10.1016/j.ibmb.2006.12.005
[26]	YOON J S, MOGILICHERLA K, GURUSAMY D, CHEN X, CHEREDDY S C, PALLI S R . Double-stranded RNA binding protein, Staufen, is required for the initiation of RNAi in coleopteran insects. Proceedings of the National Academy of Sciences of the United States of America, 2018,115(33):8334-8339. doi: 10.1073/pnas.1809381115 pmid: 30061410
[27]	XI Y, PAN P L, YE Y X, YU B, XU H J, ZHANG C X . Chitinase-like gene family in the brown planthopper, Nilaparvata lugens. Insect Molecular Biology, 2015,24(1):29-40. doi: 10.1111/imb.12133 pmid: 25224926
[28]	ZHAO L N, YANG M M, SHEN Q D, 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. Scientific Reports, 2016,6:27841. doi: 10.1038/srep27841 pmid: 27328657
[29]	LIVAK K J, SCHMITTGEN T D . Analysis of relative gene expression data using real-time quantitative PCR and the 2 ^-ΔΔCT method . Methods, 2001,25(4):402-408. doi: 10.1006/meth.2001.1262 pmid: 11846609
[30]	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 of TRE gene. Frontiers in Physiology, 2017,8:750. doi: 10.3389/fphys.2017.00750 pmid: 29033849
[31]	KANAMORI Y, SAITO A, HAGIWARA-KOMODA Y, TANAKA D, MITSUMASU K, KIKUTA S, WATANABE M, CORNETTE R, KIKAWADA T, OKUDA T . The trehalose transporter 1 gene sequence is conserved in insects and encodes proteins with different kinetic properties involved in trehalose import into peripheral tissues. Insect Biochemistry and Molecular Biology, 2010,40(1):30-37. doi: 10.1016/j.ibmb.2009.12.006
[32]	ULDRY M, THORENS B . The SLC2 family of facilitated hexose and polyol transporters. European Journal of Physiology, 2004,447(5):480-489. doi: 10.1007/s00424-003-1085-0 pmid: 12750891
[33]	SAIER M H . Genome archeology leading to the characterization and classification of transport proteins. Current Opinion in Microbiology, 1999,2(5):555-561. doi: 10.1016/s1369-5274(99)00016-8 pmid: 10508720
[34]	PRENTICE K, CHRISTIAENS O, PERTRY I, BAILEY A, NIBLETT C, GHISLAIN M, GHEYSEN G, SMAGGHE G . RNAi-based gene silencing through dsRNA injection or ingestion against the African sweet potato weevil Cylas puncticollis (Coleoptera: Brentidae). Pest Management Science, 2017,73(1):44-52. doi: 10.1002/ps.4337 pmid: 27299308
[35]	LOU Y H, PAN P L, YE Y X, CHENG C, XU H J, ZHANG C X . Identification and functional analysis of a novel chorion protein essential for egg maturation in the brown planthopper. Insect Molecular Biology, 2018,27(3):393-403. doi: 10.1111/imb.12380 pmid: 29465791
[36]	XI Y, PAN P L, ZHANG C X . The β-n-acetylhexosaminidase gene family in the brown planthopper, Nilaparvata lugens. Insect Molecular Biology, 2015,24(6):601-610. doi: 10.1111/imb.12187 pmid: 26304035
[37]	LI J X, CAO Z, GUO S, TIAN Z, LIU W, ZHU F, WANG X P . Molecular characterization and functional analysis of two trehalose transporter genes in the cabbage beetle, Colaphellus bowringi. Journal of Asia-Pacific Entomology, 2020,23(3):627-633. doi: 10.1016/j.aspen.2020.05.011

引物名称 Primer name	正向引物 Forward primer (5′-3′)	反向引物 Reverse primer (5′-3′)	产物长度 Length (bp)
dsNlTret1-like X1	GCAAACAACAGCGAGCAA	CCAAGAGGCACCCATCC	472
dsNlTret1-like X1-T7	T7-GCAAACAACAGCGAGCAA	T7-CCAAGAGGCACCCATCC	552
dsNlTret1-2 X1	CTTCGTTCACCAGCACCT	CAAATGGCACTTATTATCGTC	547
dsNlTret1-2 X1-T7	T7-CTTCGTTCACCAGCACCT	T7-CAAATGGCACTTATTATCGTC	597
dsGFP	AAGGGCGAGGAGCTGTTCACCG	CAGCAGGACCATGTGATCGCGC	657
dsGFP-T7	T7-AAGGGCGAGGAGCTGTTCACCG	T7-CAGCAGGACCATGTGATCGCGC	707
qNl18S	CGCTACTACCGATTGAA	GGAAACCTTGTTACGACTT	165
qNlTret1-like X1	GTGGGAATCGTGAACATGGG	ATGGTCATGAGTGTGCTGGA	101
qNlTret1-2 X1	TATGTGTCGGCTGGGTTCTT	CTCTGAGCCAGCACAATGAC	190
qNlTPS1	AAGACTGAGGCGAATGGT	AAGGTGGAAATGGAATGTG	154
qNlTPS2	AGAGTGGACCGCAACAACA	TCAACGCCGAGAATGACTT	161
qNlTRE1-1	GCCATTGTGGACAGGGTG	CGGTATGAACGAATAGAGCC	132
qNlTRE1-2	GATCGCACGGATGTTTA	AATGGCGTTCAAGTCAA	178
qNlTRE2	TCACGGTTGTCCAAGTCT	TGTTTCGTTTCGGCTGT	197