|
|
Cloning, Expression and Functional Analysis of Alkaline Phosphatase HcALP3 Gene in Hyphantria cunea |
ZHAO Dan1,*, CHANG Meng-Ying1,*, QIAO Ying-Cui1, WANG Zhe1, LIU Zi-Huan3, LU Xiu-Jun1,**, GUO Wei1,2,** |
1 College of Plant Protection, Hebei Agricultural University, Baoding 071001, China; 2 Graduate School of Chinese Academy of Agricultural Sciences, Beijing 100081, China; 3 Huanghua Port Customs, Cangzhou 061113, China |
|
|
Abstract The fall webworm (Hyphantria cunea) is an important quarantine pest worldwide and a major invasive pest in China. Bacillus thuringiensis (Bt) is widely used insecticidal microorganisms, however the mechanism of Bt on H. cunea is still unclear. To analyze the binding characteristics of alkaline phosphatase (ALP) of H. cunea with Bt toxin and explore the insecticidal mechanism, the HcALP3 gene (GenBank No. MH796759) was cloned by PCR method, contained a 1 557 bp ORF which encoded 518 amino acids. The recombinant HcALP3 protein induced by isopropyl-β-D-thiogalactoside (IPTG) was expressed as a 56.65 kD recombinant protein in E. coli BL21. The transcription analysis of HcALP3 gene in different tissue and at different development stages were analyzed by qRT-PCR, the results showed that HcALP3 gene was expressed in every instar and was highly abundant in the fourth-instar larvae and midgut. Ligand blot assay showed that HcALP3 protein only bound to Bt cry1Ab35 toxin specifically, could not bind to Bt Cry1Ac and Cry9Ea10. The transcript levels of HcALP3 were reduced by 75.32% using RNAi technology for gene silencing of HcALP3. The dsRNA-HcALP3 injected larvae were reared on Bt Cry1Ab35, Cry1Ac and Cry9Ea10 protein, the corrected mortalities were 45.93%, 73.33% and 70%, respectively. The results revealed that the corrected mortalities induced by Bt Cry1Ab35 to dsRNA-HcALP3 injected larvae were significantly lower than those injected with dsRNA-egfp. In this study, the HcALP3 of H. cunea was identified and the function of HcALP3 was analysed, which provides basis for further study on the mechanism of Bt against H. cunea.
|
Received: 29 September 2020
|
|
Corresponding Authors:
**luxiujun@hebau.edu.cn;guowei@hebau.edu.cn
|
About author:: **The authors who contribute equally |
|
|
|
[1] 常梦颖, 赵丹, 张雅昆, 等.2019.美国白蛾中肠碱性磷酸酶HcALP1与苏云金杆菌3种Bt蛋白的体外结合特性分析[J].蚕业科学, 45(03): 331-337. (Chang M Y, Zhao D, Zhang Y K, et al.2019.In vitro binding characteristics of Hyphantria cunea midgut HcALP1 with three kinds of Bacillus thuringiensis Cry toxins[J].Science of Sericulture, 45(03): 331-337.) [2] 陈贵东, 李燕芳, 何颖, 等.2016.鳞翅目昆虫Cry毒素受体与人同名蛋白的生物信息学分析[J].生物技术, 26(03): 272-279. (Chen G D, Li Y F, He Y, et al.2016.Bioinformatics analysis of relationship between Cry toxin receptor of lepidopteran insects and the same name protein of Homo sapiens[J].Biotechnology, 26(03): 272-279.) [3] 黄瑞芬, 马妍, 李硕, 等.2019.我国重大检疫性害虫美国白蛾生物防治研究进展[J].辽宁林业科技,(03): 42-45; 54. (Huang RF, Ma Y, Li S, et al.2019.Study progress in biological control of Hyphantria cunea-an important quarantine pest in China[J].Liaoning Forestry Science and Technology,(03): 42-45; 54.) [4] 李博.2016.二化螟ALP基因家族Cry蛋白受体功能分析[D].硕士学位论文, 华中农业大学, 导师: 雷朝亮, pp.43-50. (Li B.2016.Functional analysis of ALP gene family as the receptor of Cry proteins in Chilo suppressalis[D].Thesis for M.S., Huazhong Agricultural University, Supervisor: Lei C L, pp.43-50.) [5] 刘云鹏, 张啸天, 解春霞, 等.2019.美国白蛾高毒Bt菌株复配增效试验[J].安徽农业大学学报, 46(05): 870-875. (Liu Y P, Zhang X T, Xie C X, et al.2019.Synergistic test of Bt strain with high virulence of Hyphantria cunea[J].Journal of Anhui Agricultural University, 46(05): 870-875.) [6] 李文楚.2004.软化病感染家蚕的碱性磷酸酶活力测定及病理学研究[J].华南农业大学学报, 25(4): 120-122. (Li W C.2004.Studies on the activities of alkaline phosphatase and pathology of Bombyx mori infected with flacherie[J].Journal of South China Agricultural University, 25(4): 120-122.) [7] 林莉.2019.苏云金杆菌Cry1A毒素与褐飞虱中肠内潜在受体互作分析[D].博士学位论文, 福建农林大学, 导师: 关雄; 绍恩斯, pp.58-79. (Lin L.2019.Interaction of Bacillus thuringiensis Cry1A toxin and candidate receptors in the midgut of Nilaparvata lugens (Stal)[D].Thesis for Ph.D., Fujian Agriculture and Forestry university, Supervisor: Guan X; Shao En S, pp.58-79.) [8] 彭琦, 周子珊, 张杰.2015.苏云金芽胞杆菌杀虫晶体蛋白研究进展[J].中国生物防治学报, 31(05): 712-722. (Peng Q, Zhou Z S, Zhang J.2015.Research prospects in insecticidal crystal proteins of Bacillus thuringiensis[J].Chinese Journal of Biological Control, 31(05): 712-722.) [9] 谢菲, 张人伟, 李翼, 等.2019.浅谈美国白蛾的监测与防治[J].南方林业科学, 47(04):60-61, 64. (Xie F, Zhang R W, Li Y, et al.2019.Remark on the monitor and prevention of Hyphantria cunea[J].South China Forestry Science, 47(04): 60-61, 64.) [10] 袁向东, 张万娜, 赵曼, 等.2017.甜菜夜蛾中肠碱性磷酸酶alp2基因的克隆、表达及功能分析[J].植物保护学报, 44(01): 8-15. (Yuan X D, Zhang W N, Zhao M, et al.2017.Cloning, expression and functional analysis of alkaline phosphatase 2 (alp2) in the midgut of beet armyworm Spodoptera exigua (Hübner)(Lepidoptera: Noctuidae)[J].Journal of Plant Protection, 44(01): 8-15.) [11] 张霄, 胡晓丹, 仲建锋,等.2016.小菜蛾碱性磷酸酯酶受体表达与分子模拟[J].中国农业科学, 49(23): 4555-4565. (Zhan Xiao, Hu X D, Zhong J F, et al.2016.Expression and molecular simulation of alkaline phosphatase receptor of Plutella xyllostella[J].Scientia Agricultura Sinica, 49(23): 4555-4565.) [12] 张涛, 张丽丽, 魏纪珍, 等.2013.Cry1Ac抗、感棉铃虫碱性磷酸酯酶(ALP1)的表达量比较[J].中国农业科学, 46(17): 3580-3586. (Zhang T, Zhang L L, Wei J Z, et al.2013.The Expression level of alkaline phosphatase (ALP1) in Cry1Ac-resistant and susceptible cotton bollworm Helicoverpa armigera Hübner[J].Scientia Agricultura Sinica, 46(17): 3580-3586.) [13] 张雅昆.2018.美国白蛾中肠氨肽酶N的鉴定及其在Cry1Ab35蛋白杀虫机制中功能研究[D].博士学位论文, 河北农业大学, 导师: 郭巍, pp.55-70. (Zhang Y K.2018.Identificaction and functional characteration of Hyphantria cunea aminopeptidase N in Cry1Ab35 intoxication[D].Thesis for Ph.D., Hebei Agricultural University, Suppervisor: Guo W, pp.55-70.) [14] Adang M J, Crickmore N, Jurat-Fuentes J L.2014.Advances in insect physiology: Chapter 2-diversity of Bacillus thuringiensis crystal toxins and mechanism of action[M].San Diego: Elsevier, 47, 39-87. [15] Arenas I, Bravo A, Soberon M, et al.2010.Role of alkaline phosphatase from Manduca sexta in the mechanism of action of Bacillus thuringiensis Cry1Ab toxin[J].The Journal of Biological Chemistry, 285(17): 12497-12503. [16] Bai P H, Wang H M, Liu B S, et al.2020.Botanical volatiles selection in mediating electrophysiological responses and reproductive behaviors for the fall webworm moth Hyphantria cunea[J].Frontiers in Physiology, 11: 486 [17] Cook B J, Nelson D R, Hipps P.1969.Esterases and phosphatases in the gastric secretion of the cockroach, Periplanetaamericana[J].Journal of Insect Physiology, 15(4): 581-589. [18] Chen J, Aimanova K, Gill S S.2017.Functional characterization of Aedes aegypti alkaline phosphatase ALP1 involved in the toxicity of Cry toxins from Bacillus thuringiensis subsp.israelensis and jegathesan[J].Peptides, 98: 78-85. [19] Fernando Z, Isabel G, Guadalupe P, et al.2013.A tenebrio molitor GPI-anchored alkaline phosphatase is involved in binding of Bacillus thuringiensis Cry3Aa to brush border membrane vesicles[J].Peptides, 41: 81-86. [20] Hua G, Zhang R, Bayyareddy K, et al.2009.Anopheles gambiae alkalinephosphatase is a functional receptor of Bacillus thuringiensis jegathesan Cry11Ba toxin[J].Biochemistry, 48(41): 9785-9793. [21] Gulnar G, Mehmet M, Zulfi M.2020.Biological characteristics and efficacy of Bacillus thuringiensis var.thuringiensis against the cotton leaf roller, Syllepte derogata (Fabricius, 1775)(Lepidoptera: Crambidae)[J].Egyptian Journal of Biological Pest Control, 30(1): 265-267. [22] Jimenez A I, Reyes E Z, Cancino-RodeznoA, et al.2012.Aedes aegypti alkaline phosphatase ALP1 is a functional receptor of Bacillus thuringiensis Cry4Ba and Cry11Aa toxins[J].Insect Biochemistry and Molecular Biology, 42(9): 683-689. [23] Jurat-Fuentes Juan Luis, Karumbaiah Lohitash, Jakka Siva Rama Krishna, et al.2011.Reduced levels of membrane-bound alkaline phosphatase are common to lepidopteran strains resistant to Cry toxins from Bacillus thuringiensis[J].PLOS ONE, 6(3): e17606. [24] Jakka S R K, Gong L, Hasler J, et al.2016.Field-evolved mode 1 resistance of the fall armyworm to transgenic Cry1Fa-expressing corn associated with reduced Cry1Fa toxin binding and midgut alkaline phosphatase expression[J].Applied and Environmental Microbiology, 82(4): 1023-1034. [25] Melo A L, Soccol V T, Soccol C R.2016.Bacillus thuringiensis: Mechanism of action, resistance, and new applications: A review[J].Critical Reviews In Biotechnology, 36(2): 317-26. [26] Miranda R, Zamudio F Z, Bravo A.2001.Processing of Cry1Ab delta-endotoxin from Bacillus thuringiensis by Manduca sexta and Spodoptera frugiperda midgut proteases: Role in protoxin activation and toxin inactivation[J].Insect Biochem and Molecular Biology, 31(12): 1155-1163. [27] Pan Z Z,Xu L,Liu B,et al.2017.PxAPN5 serves as a functional receptor of Cry2Ab in Plutellaxylostella (L.) and its binding domain analysis[J].International Journal of Biological Macromolecules, 105: 516-521. [28] Qiu L, Wang P P, Zhang B Y, et al.2017.Proteomic analysis of Cry1Ac and Cry2Aa binding proteins in brush border membrane vesicles of the Chilo suppressalis midgut[J].Journal of Asia-Pacific Entomology, 20: 1165-1169. [29] Wang J, Zhang H N, Wang HD, et al.2016.Functional validation of cadherin as a receptor of Bt toxin Cry1Ac in Helicoverpa armigera utilizing the CRISPR/Cas9 system[J].Insect Biochemistry and Molecular Biology, 76: 11-17. [30] Wei J, Zhang M, Liang G, Li X.2019.Alkaline phosphatase 2 is a functional receptor of Cry1Ac but not Cry2Ab in Helicoverpa zea[J].Insect Biochem and Molecular Biology, 28(3): 372-379. [31] Yuan X D, Zhao M, Wei J Z, et al.2017.New insights on the role of alkaline phosphatase 2 from Spodoptera exigua (Hübner) in the action mechanism of Bt toxin Cry2Aa[J].Journal of Insect Physiology, 98: 101-107. [32] Zhang Y K, Zhao D, Yan X P, et al.2017.Identification and characterization of Hyphantria cunea aminopeptidase N as a binding protein of Bacillus thuringiensis Cry1Ab35 toxin[J].International Journal of Molecular Sciences, 18(12): 2575. |
|
|
|