半胱氨酸蛋白酶是植食性昆虫体不可或缺的重要水解蛋白酶之一。本文从小贯小绿叶蝉的转录组数据库中获得半胱氨酸蛋白酶基因Eocyp的基因片段的转录本序列,利用RACE技术得到其cDNA全长序列,利用生物信息学技术对Eocyp所编码的理论氨基酸序列进行分析,使用qRT-PCR技术检测Eocyp在小贯小绿叶蝉不同发育阶段,雌、雄成虫不同部位以及不同温度和光周期下的表达量。结果表明,小贯小绿叶蝉Eocyp的全长cDNA序列包含一个1β656βbp的开放阅读框,编码551个氨基酸,并具有由24个氨基酸残基组成的信号肽。推测其相对分子量为62.09βkD,预测其分子式为C2782H4163N737O835S25;理论等电点为6.02,不稳定系数为36.10。Eocyp具有半胱氨酸蛋白酶基因的典型特征,即催化三联体Cys357-His499-Asn519,同时具有ERFNIN,GNFD和GCDGG簇等保守结构域。Eocyp编码氨基酸序列与茶翅蝽CYP基因编码序列相似度最高。表达特征分析结果表明,Eocyp在小贯小绿叶蝉的各个生长时期均有表达,在5龄若虫时期达到最高;Eocyp在小贯小绿叶蝉雌、雄成虫的腹部均有较高的表达量;然而,Eocyp的表达量对于高、低温和不同光周期均无响应。这些结果为进一步研究Eocyp功能提供了理论依据。
Cysteine proteinases are important proteolytic enzymes for herbivorous insects. In this paper, the transcript sequence of Eocyp was obtained from the transcriptome database of Empoasca onukii. The full-length cDNA sequence of Eocyp was then cloned by RACE. The theoretical amino acid sequence of Eocyp was analyzed by bioinformatics. The expression levels of Eocyp in different developmental stages, different parts of female and male adults of E. onukii and nymphs under different temperatures and photoperiods were detected by qRT-PCR. The full-length cDNA sequence of Eocyp contained an open reading frame of 1β656βbp, which encoded 551 amino acids and had a signal peptide consisting of 24 amino acid residues. Its molecular formula was predicted to be C2782H4163N737O835S25. Its relative molecular weight and the theoretical isoelectric point were estimated to be 62.09βkD and 6.02, and the instability coefficient of Eocyp was 36.10. Eocyp had the typical feature of cysteine protease, a catalytic triad Cys357-His499-Asn519, and the conserved domains such as ERFNIN, GNFD amino acid residues and the GCDGG clusters. The amino acid sequence of Eocyp had a high similarity with the CYP gene of Halyomorpha halys. Expression analysis showed that Eocyp was expressed in all developmental stages of E. onukii with its peak in the fifth instar nymphs. Higher expression of Eocyp was observed in the abdomen of adult E. onukii, irrespective of female and male. However, the expression level of Eocyp did not respond to the variation of temperatures and photoperiods. These results provided a theoretical basis for further study of Eocyp.
[1] Berti P J, Storer A C.Alignment/phylogeny of the papain superfamily of cysteine proteases[J]. Journal of Molecular Biology, 1995, 246(2): 273-283.
[2] Chernaya V I.Early Postnatal Development-related dynamics of the activity of cathepsin l in rat brain structures[J]. Neurophysiology, 2001, 33(1): 15-18.
[3] Barros N M, Puzer L, Tersariol I L, et al.Plasma prekallikrein/kallikrein processing by lysosomal cysteine proteases[J]. Biological Chemistry, 2004, 385(11): 1087-1091.
[4] Cristofoletti P T, Ribeiro A F, Deraison C, et al.Midgut adaptation and digestive enzyme distribution in a phloem feeding insect, the pea aphid Acyrthosiphon pisum[J]. Journal of Insect Physiology, 2003, 49(1): 11-24.
[5] Rabossi A, Stoka V, Puizdar V, et al.Novel aspartyl proteinase associated to fat body histolysis during Ceratitis capitata early metamorphosis[J]. Archives of Insect Biochemistry & Physiology, 2004, 57(2): 51-67.
[6] Zhao X, An X, Wang J, et al.Expression of the Helicoverpa cathepsin b-like proteinase during embryonic development[J]. Arch Insect Biochem Physiol, 2005, 58(1): 39-46.
[7] Liu J, Shi G P, Zhang W Q, et al.Cathepsin L function in insect moulting: molecular cloning and functional analysis in cotton bollworm, Helicoverpa armigera[J]. Insect Molecular Biology, 2006, 15(6): 823-834.
[8] Beton D, Guzzo C R, Ribeiro A F, et al.The 3D structure and function of digestive cathepsin L-like proteinases of Tenebrio molitor larval midgut[J]. Insect Biochemistry & Molecular Biology, 2012, 42(9): 655-664.
[9] L F Wang, L Q Chai, He H J, et al. A cathepsin L-like proteinase is involved in moulting and metamorphosis in Helicoverpa armigera[J]. Insect Molecular Biology, 2010, 19(1): 99-111.
[10] Kwangsik L, Boyeon K, Youngmoo C, et al.Expression profile of cathepsin B in the fat body of Bombyx mori during metamorphosis[J]. Comparative Biochemistry & Physiology Part B Biochemistry & Molecular Biology, 2009, 154(2): 188-194.
[11] Yu J, Wu F Y, Zou F M, et al.Identification of ecdysone response elements (EcREs) in the Bombyx mori cathepsin D promoter[J]. Biochemical & Biophysical Research Communications, 2012, 425(1): 113-118.
[12] Cai X Y, Yu J, Yu H Y, et al.Core promoter regulates the expression of cathepsin B gene in the fat body of Bombyx mori[J]. Gene, 2014, 542(2): 232-239.
[13] 苏晶晶, 陈思源, 张奎, 等. 家蚕组织蛋白酶O (BmCatO) 基因鉴定及表达分析[J]. 中国农业科学, 2015, 48(22): 4564-4573.
[14] Wu F, Kang L, Wang P, et al.The expression analysis of cysteine proteinase-like protein in wild-type and nm2 mutant silkworm (Lepidoptera: Bombyx mori)[J]. Gene, 2016, 586(1): 170-175.
[15] Peter M, Turnšek N, Gašparič MB, et al.A complex of genes involved in adaptation of Leptinotarsa decemlineata larvae to induced potato defense[J]. Arch Insect Biochem Physiol, 2012, 79(3): 153-181.
[16] 赵冬香, 陈宗懋, 程家安. 茶小绿叶蝉优势种的归属[J]. 茶叶科学, 2000, 20(2): 101-104.
[17] 王念武, 徐金汉, 陈峥, 等. 不同茶园假眼小绿叶蝉抗药性比较[J]. 福建农业大学学报, 2004(2): 169-173.
[18] 庄家祥, 傅建炜, 苏庆泉, 等. 福建省茶小绿叶蝉抗药性的地区差异[J]. 茶叶科学, 2009, 29(2): 154-158.
[19] Bolter C, Jongsma M A.The adaptation of insect to plant protease inhibitors[J]. Journal of Insect Physiology. 1997, 43(10): 885-895.
[20] 杨文佳, 许抗抗, 王进军, 等. 橘小实蝇半胱氨酸蛋白酶基因克隆及表达模式[J]. 中山大学学报(自然科学版), 2017, 56(1): 131-137.
[21] 宋旺. 花绒寄甲Caspase-1基因的表达研究[D]. 杨凌: 西北农林科技大学, 2014.
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