绿盲蝽雷帕霉素靶蛋白的克隆、抗体制备及在蜕皮激素诱导下的应答

doi:10.3864/j.issn.0578-1752.2021.10.008

摘要/Abstract

摘要：

【目的】克隆绿盲蝽（Apolygus lucorum）雷帕霉素靶标全长基因（AlTOR）,研究AlTOR时空表达特性及在外源蜕皮激素（20E）诱导下的应答响应,进一步获得原核表达的重组蛋白及制备多克隆抗体,分析该抗体的特异性,为后续研究AlTOR蛋白功能打下基础。【方法】RACE法克隆AlTOR基因序列全长,qRT-PCR分析AlTOR表达特性以及在20E及其抑制剂U73122诱导下的应答响应;将含有AlTOR序列T载体经EcoR I及Xho I双酶切,构建原核表达载体pCzn1-AlTOR,经20℃、0.5 mmol·L^-1 IPTG诱导表达、变性、复性和蛋白纯化后,以获得AlTOR重组蛋白,最后再通过免疫,制备AlTOR蛋白多克隆抗体,Western blot评价该抗体的特异性。【结果】AlTOR的开放阅读框编码包含435个氨基酸,具有典型的TOR基因序列特征,即SIN1结构域、高度保守的CRIM结构域及PH域;AlTOR在绿盲蝽不同日龄、不同组织中均有表达,且在1日龄以及脂肪体中表达量最高;20E可诱导绿盲蝽不同日龄若虫期AlTOR的表达,同时也能诱导成虫头、翅、卵巢和脂肪体中AlTOR表达上调,分别上调200.00%、118.89%、20.53%和60.82%,而U73122在中肠和卵巢中会显著抑制AlTOR的表达。经双酶切的重组克隆载体可成功亚克隆到pCzn1载体上,命名为pCzn1-AlTOR;IPTG诱导的重组质粒可特异性表达一个约36 kD的蛋白,且主要以包涵体形式存在;经Ni-IDA亲和层析纯化后,仅在36 kD附近有一条明显的特异性条带。进一步免疫及间接ELISA方法确定抗血清对TOR蛋白的效价,Western blot分析表明制备的多克隆抗体可以特异性与AlTOR重组蛋白及绿盲蝽3龄若虫总蛋白结合,说明制备的AlTOR多克隆抗体特异性较好,可以用于后续的蛋白研究。【结论】AlTOR在绿盲蝽体内的表达谱显示出发育阶段特异性和组织特异性;20E及其抑制剂诱导后,AlTOR呈现出相反的应答反应;本研究获得的AlTOR重组蛋白的多克隆抗体具有高特异性。

关键词: 绿盲蝽, 雷帕霉素靶标, 基因克隆, 蜕皮激素, 多克隆抗体, 表达

Abstract:

【Objective】The objective of this study is to clone the target of rapamycin gene (TOR) of Apolygus lucorum (AlTOR), analyze the expression profile of AlTOR as well as its expression patterns after injection of 20-hydroxyecdysone (20E) and its inhibitor U73122, obtain the recombinant protein and antibody against AlTOR protein, and to lay the foundation for the further study of AlTOR protein function.【Method】The full-length of AlTOR was cloned by RACE method. Using qRT-PCR technique, the expression pattern of AlTOR was determined at different developmental stages and different tissues in A. lucorum, as well as its expression patterns after injection of 20E and U73122. To construct its prokaryotic expression pCzn1-AlTOR, T vector containing the target gene was digested with the restriction enzyme EcoR I and Xho I and subcloned. The recombinant plasmid containing target gene was specifically expressed after induction by IPTG. The target recombinant protein has over expressed and has been purified by using Ni-NTA agarose. The polyclonal antibody was obtained by rabbit immunity, cell fusion and ascites preparation, and Western blot was used to detect the specificity of antibody.【Result】The open reading frame of AlTOR encodes 435 amino acids. AlTOR protein has the typical domains including the SIN1 domain, highly conserved CRIM domain and PH domain. qRT-PCR results showed that AlTOR was expressed in 1-day-old to 16-day-old of A. lucorum, in which the expression level was the highest at 1-day-old. AlTOR was expressed in all tissues of A. lucorum female adults, and the fat body had the highest expression level. 20E could induce the expression of AlTOR at different day-old nymphs, and could also induce the up-regulated expression of AlTOR in the head, wing, ovary and fat body of the female adults, which was increased by 200.00%, 118.89%, 20.53% and 60.82%, respectively, while U73122 significantly suppressed AlTOR expression in the midgut and ovary. The vector containing AlTOR was digested and linked to the vector pCzn1. The recombinant plasmid pCzn1-AlTOR expressed the target recombinant protein of 36 kD after induction by IPTG. The 36 kD target protein from the strain containing AlTOR was obtained by the Ni-NTA agarose and used as the antigen, and one cell line polyclonal antibody could specifically bind to the AlTOR recombinant protein and the total protein of the 3rd instar nymph of A. lucorum, indicating that the prepared AlTOR polyclonal antibody has good specificity.【Conclusion】The expression of AlTOR shows the developmental stage- and tissue-specificity. After the induction of 20E and its inhibitor, AlTOR shows the opposite responses. The obtained polyclonal antibody against AlTOR recombinant protein is highly specific.

Key words: Apolygus lucorum, target of rapamycin (TOR), gene cloning, 20-hydroxyecdysone (20E), polyclonal antibody, expression

谭永安,赵旭东,姜义平,赵静,肖留斌,郝德君. 绿盲蝽雷帕霉素靶蛋白的克隆、抗体制备及在蜕皮激素诱导下的应答[J]. 中国农业科学, 2021, 54(10): 2118-2131.

TAN YongAn,ZHAO XuDong,JIANG YiPing,ZHAO Jing,XIAO LiuBin,HAO DeJun. Cloning, Preparation of Antibody and Response Induced by 20-Hydroxyecdysone of Target of Rapamycin in Apolygus lucorum[J]. Scientia Agricultura Sinica, 2021, 54(10): 2118-2131.

图/表 11

表1

图1

图2

图3

图4

图5

图6

图7

图8

图9

图10

参考文献 41

[1]	LU Y H, WU K M, JIANG Y Y, XIA B, LI P, FENG H Q, WYCKHUYS K A G, GUO Y Y. Mirid bug outbreaks in multiple crops correlated with wide-scale adoption of Bt cotton in China. Science, 2010,328(5982):1151-1154. doi: 10.1126/science.1187881
[2]	WU K M, MU W, LIANG G M, GUO Y Y. Regional reversion of insecticide resistance in Helicoverpa armigera (Lepidoptera: Noctuidae) is associated with the use of Bt cotton in northern China. Pest Management Science, 2005,61(5):491-498. doi: 10.1002/(ISSN)1526-4998
[3]	陆宴辉, 梁革梅, 吴孔明. 棉盲蝽综合治理研究进展. 植物保护, 2007,33(6):10-15.
	LU Y H, LIANG G M, WU K M. Advances in integrated management of cotton mirids. Plant Protection, 2007,33(6):10-15. (in Chinese)
[4]	LU Y H, WU K M, JIANG Y Y, GUO Y Y, DESNEUX N. Widespread adoption of Bt cotton and insecticide decrease promotes biocontrol services. Nature, 2012,487(7407):362-365. doi: 10.1038/nature11153
[5]	NELSEN C J, RICKHEIM D G, TUCKER M M, HANSEN L K, ALBRECHT J H. Evidence that cyclin D1 mediates both growth and proliferation downstream of TOR in hepatocytes. The Journal of Biological Chemistry, 2003,278(6):3656-3663. doi: 10.1074/jbc.M209374200
[6]	DE VIRGILIO C, LOEWITH R. The TOR signalling network from yeast to man. The International Journal of Biochemistry and Cell Biology, 2006,38(9):1476-1481. doi: 10.1016/j.biocel.2006.02.013
[7]	AVRUCH J, LONG X M, LIN Y, ORTIZ-VEGA S, RAPLEY J, PAPAGEORGIOU A, OSHIRO N, KIKKAWA U. Activation of mTORC1 in two steps: Rheb-GTP activation of catalytic function and increased binding of substrates to raptor1. Biochemical Society Transactions, 2009,37(1):223-226. doi: 10.1042/BST0370223
[8]	刘南南, 姚军虎. 营养素和激素对乳蛋白合成过程中哺乳动物雷帕霉素靶蛋白信号通路调节作用的研究进展. 动物营养学报, 2013,25(6):1158-1163.
	LIU N N, YAO J H. Research advance in regulation of mTOR signaling pathway by nutrients and hormones in milk protein synthesis. Chinese Journal of Animal Nutrition, 2013,25(6):1158-1163. (in Chinese)
[9]	LIU L, LUO Y, CHEN L, SHEN T, XU B S, CHEN W X, ZHOU H Y, HAN X Z, HUANG S L. Rapamycin inhibits cytoskeleton reorganization and cell motility by suppressing RhoA expression and activity. The Journal of Biological Chemistry, 2010,285(49):38362-38373. doi: 10.1074/jbc.M110.141168
[10]	LIN X Y, SMAGGHE G. Roles of the insulin signaling pathway in insect development and organ growth. Peptides, 2019,122:169923. doi: 10.1016/j.peptides.2018.02.001
[11]	LIN X Y, DE SCHUTTER K, CHAFINO S, FRANCH-MARRO X, MARTIN D, SMAGGHE G. Target of rapamycin (TOR) determines appendage size during pupa formation of the red flour beetle Tribolium castaneum. Journal of Insect Physiology, 2019,117:103902. doi: 10.1016/j.jinsphys.2019.103902
[12]	LAYALLE S, ARQUIER N, LEOPOLD P. The TOR pathway couples nutrition and developmental timing in Drosophila. Developmental Cell, 2008, 15(4):568-577. doi: 10.1016/j.devcel.2008.08.003
[13]	OKAMOTO N, YAMANAKA N, YAGI Y, NISHIDA Y, KATAOKA H, O’CONNOR M B, MIZOGUCHI A. A fat body-derived IGF-like peptide regulates postfeeding growth in Drosophila. Developmental Cell, 2009,17(6):885-891. doi: 10.1016/j.devcel.2009.10.008
[14]	DELANOUE R, SLAIDINA M, LEOPOLD P. The steroid hormone ecdysone controls systemic growth by repressing dMyc function in Drosophila fat cells. Developmental Cell, 2010,18(6):1012-1021. doi: 10.1016/j.devcel.2010.05.007
[15]	COLOMBANI J, RAISIN S, PANTALACCI S, RADIMERSKI T, MONTAGNE J, LEOPOLD P. A nutrient sensor mechanism controls Drosophila growth. Cell, 2003,114(6):739-749. doi: 10.1016/S0092-8674(03)00713-X
[16]	COLOMBANI J, BIANCHINI L, LAYALLE S, PONDEVILLE E, DAUPHIN-VILLEMANT C, ANTONIEWSKI C, CARRE C, NOSELLI S, LEOPOLD P. Antagonistic actions of ecdysone and insulins determine final size in Drosophila. Science, 2005,310(5748):667-670. doi: 10.1126/science.1119432
[17]	MENAND B, DESNOS T, NUSSAUME L, BERGER F, BOUCHEZ D, MEYER C, ROBAGLIA C. Expression and disruption of the Arabidopsis TOR (target of rapamycin) gene. Proceedings of the National Academy of Sciences of the United States of America, 2002,99(9):6422-6427.
[18]	DAMES S A, MULET J M, RATHGEB-SZABO K, HALL M N, GRZESIEK S. The solution structure of the FATC domain of the protein kinase target of rapamycin suggests a role for redox-dependent structural and cellular stability. The Journal of Biological Chemistry, 2005,280(21):20558-20564. doi: 10.1074/jbc.M501116200
[19]	宋晓丹, 张园, 邹祥. TOR信号调控真菌细胞的生长与代谢. 微生物学报, 2018,58(10):1691-1700.
	SONG X D, ZHANG Y, ZOU X. Fungal cell growth and metabolism regulated by the TOR signal pathway. Acta Microbiologica Sinica, 2018,58(10):1691-1700. (in Chinese)
[20]	SUN Y, XIAO L B, CAO G C, ZHANG Y J, XIAO Y F, XU G C, ZHAO J, TAN Y A, BAI L X. Molecular characterisation of the vitellogenin gene (AlVg) and its expression after Apolygus lucorum had fed on different hosts. Pest Management Science, 2016,72(9):1743-1751. doi: 10.1002/ps.4203
[21]	KLEIN R R, BOURDON D M, COSTALES C L, WAGNER C D, WHITE W L, WILLIAMS J D, HICKS S N, SONDEK J, THAKKER D R. Direct activation of human phospholipase C by its well known inhibitor U73122. Journal of Biological Chemistry, 2011,286(14):12407-12416. doi: 10.1074/jbc.M110.191783
[22]	SONG N N, DING W H, CHU S Y, ZHAO J, DONG X, DI B B, TANG C S. Urotensin II stimulates vascular endothelial growth factor secretion from adventitial fibroblasts in synergy with angiotensin II. Circulation Journal, 2012,76(5):1267-1273. doi: 10.1253/circj.CJ-11-0870
[23]	LIVAK K J, SCHMITTGEN T D. Analysis of relative gene expression data using real-time quantitative PCR and the 2^-ΔΔCT method. Methods, 2001,25:402-408. doi: 10.1006/meth.2001.1262
[24]	LUSHCHAK O, STRILBYTSKA O, PISKOVATSKA V, STOREY K B, KOLIADA A, VAISERMAN A. The role of the TOR pathway in mediating the link between nutrition and longevity. Mechanisms of Ageing and Development, 2017,164:127-138. doi: 10.1016/j.mad.2017.03.005
[25]	YERLIKAYA S, MEUSBURGER M, KUMARI R, HUBER A, ANRATHER D, COSTANZO M, BOONE C, AMMERER G, BARANOV P V, LOEWITH R. TORC1 and TORC2 collude to regulate Ribosomal Protein S6 phosphorylation in Saccharomyces cerevisiae. Molecular Biology of the Cell, 2016,27(2):397-409. doi: 10.1091/mbc.e15-08-0594
[26]	AVRUCH J, PRASKOVA M, ORTIZ-VEGA S, LIU M, ZHANG X F. Nore1 and RASSF1 regulation of cell proliferation and of the MST1/2 kinases//Methods in Enzymology, 2006,407:290-310.
[27]	LOEWITH R, HALL M N. Target of rapamycin (TOR) in nutrient signaling and growth control. Genetics, 2011,189(4):1177-1201. doi: 10.1534/genetics.111.133363
[28]	ZHA X J, SUN Q, ZHANG H B. mTOR upregulation of glycolytic enzymes promotes tumor development. Cell Cycle, 2011,10(7):1015-1016. doi: 10.4161/cc.10.7.15063
[29]	RAJAN A, PERRIMON N. Drosophila cytokine unpaired 2 regulates physiological homeostasis by remotely controlling insulin secretion. Cell, 2012,151(1):123-137. doi: 10.1016/j.cell.2012.08.019
[30]	FENG Y H, WU L S. mTOR up-regulation of PFKFB3 is essential for acute myeloid leukemia cell survival. Biochemical and Biophysical Research Communication, 2017,483(2):897-903. doi: 10.1016/j.bbrc.2017.01.031
[31]	GHOSH D, SRIVASTAVA G P, XU D, SCHULZ L C, ROBERTS R M. A link between SIN1 (MAPKAP1) and poly (rC) binding protein 2 (PCBP2) in counteracting environmental stress. Proceedings of the National Academy of Sciences of the United States of America, 2008,105(33):11673-11678.
[32]	SMYKAL V, RAIKHEL A S. Nutritional control of insect reproduction. Current Opinion in Insect Science, 2015,11:31-38. doi: 10.1016/j.cois.2015.08.003
[33]	DANIELSEN E T, MOELLER M E, REVITZ K F. Nutrient signaling and developmental timing of maturation. Current Topics in Developmental Biology, 2013,105(3):37-67.
[34]	CARPENTER V K, DRAKE L L, AGUIRRE S E, PRICE D P, RODRIGUEZ S D, HANSEN I A. SLC7 amino acid transporters of the yellow fever mosquito Aedes aegypti and their role in fat body TOR signaling and reproduction. Journal of Insect Physiology, 2012,58(4):513-522. doi: 10.1016/j.jinsphys.2012.01.005
[35]	LU K, CHEN X, LIU W T, ZHOU Q. TOR pathway-mediated juvenile hormone synthesis regulates nutrient-dependent female reproduction in Nilaparvata lugens (Stål). International Journal of Molecular Sciences, 2016,17(4):438. doi: 10.3390/ijms17040438
[36]	KOYAMA T, MENDES C C, MIRTH C K. Mechanisms regulating nutrition-dependent developmental plasticity through organ-specific effects in insects. Frontiers in Physiology, 2013,4:263.
[37]	KADAMUR G, ROSS E M. Mammalian phospholipase C. Annual Review of Physiology, 2013,75:127-154. doi: 10.1146/annurev-physiol-030212-183750
[38]	GU S H, CHEN C H, LIN P L, HSIEH H Y. Role of protein phosphatase 2A in PTTH-stimulated prothoracic glands of the silkworm, Bombyx mori. General and Comparative Endocrinology, 2019,274:97-105. doi: 10.1016/j.ygcen.2019.01.009
[39]	GU S H, HSIEH Y C, LIN P L. Signaling involved in PTTH- stimulated 4E-BP phosphorylation in prothoracic gland cells of Bombyx mori. Journal of Insect Physiology, 2017,96:1-8. doi: 10.1016/j.jinsphys.2016.10.007
[40]	SCHLUEPMANN H, PELLNY T, VAN DIJKEN A, SEMMKENS S, PAUL M. Trehalose 6-phosphate is indispensable for carbohydrate utilization and growth in Arabidopsis thaliana. Proceedings of the National Academy of Sciences of the United States of America, 2003,100(11):6849-6854.
[41]	谭永安, 肖留斌, 孙洋, 柏立新. 绿盲蝽水溶性海藻糖酶ALTre-1基因原核表达、纯化与酶学特性. 中国农业科学, 2013,46(17):3587-3593.
	TAN Y A, XIAO L B, SUN Y, BAI L X. Prokaryotic expression, purification and functional activity assay in vitro of soluble trehalse from Apolygus lucorum. Scientia Agricultura Sinica, 2013,46(17):3587-3593. (in Chinese)

引物名称/目的 Primer name/Purpose	序列 Primer sequence (5′ to 3′)
克隆Cloning
5′-AlTOR-1	AACGACTGGGCAAGAA
5′-AlTOR-2	CAAGACGTTTGGGTAGCA
3′-AlTOR-1	AGAGACTCAGTCGCTCACTATCAAGA
3′-AlTOR-2	CATTGAGACCACACCCCAGACACA
AlTOR-F	ATGGTGGAAAAATATGCTCG
AlTOR-R	GCTAGCCAGTATACAACG
表达谱分析qRT-PCR
RT-AlTOR-F	GGGGTCGTCAACTGGCAGAA
RT-AlTOR-R	CCTCCTTCTTCTTGGGTGCGA
Al-β-Actin-F	ACCTGTACGCCAACACCGT
Al-β-Actin-R	TGGAGAGAGAGGCGAGGAT