大草蛉雄虫卵黄原蛋白基因的鉴定和表达特征

doi:10.16409/j.cnki.2095-039x.2020.04.001

摘要/Abstract

摘要： 为探索卵黄原蛋白基因对大草蛉雄虫生殖生理等活动的调节作用，利用PCR技术克隆了大草蛉卵黄原蛋白基因部分cDNA序列，采用生物信息学方法分析其序列特征，通过实时荧光定量PCR技术检测了该基因在不同发育阶段和不同组织的表达特征。结果表明，克隆到的卵黄原蛋白基因的cDNA长度为1501 bp，编码501个氨基酸，其预测的蛋白质分子量为206 kD，等电点8.11，具有vitellogenin保守的半胱氨基酸残基、GL/ICG、RXXR等结构域。序列分析表明大草蛉vitellogenin与其他昆虫同源蛋白的氨基酸序列一致性不太高，在29.46%～36.43%，与骚扰锥蝽在系统进化上关系最近。荧光定量PCR结果显示vitellogenin在不同日龄的成虫中表达量没有显著差异；在雄虫的腹部表达量较高，均高于头部和胸部，且头部和胸部的表达量之间没有显著差异，表达量均较低。结果表明vitellogenin基因在大草蛉中的表达具有组织的特异性，推测其可能参与大草蛉雄虫生殖调控。

关键词: 大草蛉, 卵黄原蛋白基因, 克隆, 表达

Abstract: In order to explore the regulatory effect of vitellogenin gene on the reproductive physiology and other activities of male Chrysopa pallens, the partial sequence of vitellogenin gene was cloned by PCR, the sequence characteristics were analyzed by bioinformatics, and the expression characteristics of vitellogenin gene in different developmental stages and different tissues were detected by real-time fluorescence quantitative PCR. The results showed that the cDNA length of the cloned vitellogenin gene was 1501 bp, encoding 501 amino acids, the predicted molecular weight of the protein was 206 kD, the isoelectric point was 8.11, and it had vitellogenin conserved cysteine residues, GL/ICG, RXXR and other domains. Sequence analysis showed that the amino acid sequence of C. pallens vitellogenin was not very consistent with that of other insect homologous proteins, which was about 29.46% and 36.43%, which was closely related to the phylogeny of Triatoma infestans. The results of fluorescence quantitative PCR showed that there was no significant difference in the expression of vitellogenin in different ages of adults, but the expression in the abdomen of males was higher than that in the head and chest, and there was no significant difference between the head and chest, but the expression was low. The results showed that the expression of vitellogenin gene in C. pallens was tissue-specific, and it was speculated that it might be involved in the reproductive regulation of male of C. pallens.

Key words: Chrysopa pallens, vitellogenin gene, cloning, expression

中图分类号:

S476.2

冯彦姣, 刘小平, 张婷婷, 陈红印, 王孟卿, 刘晨曦, 李玉艳, 张礼生, 毛建军. 大草蛉雄虫卵黄原蛋白基因的鉴定和表达特征[J]. 中国生物防治学报, 2021, 37(1): 77-85.

FENG Yanjiao, LIU Xiaoping, ZHANG Tingting, CHEN Hongyin, WANG Mengqing, LIU Chenxi, LI Yuyan, ZHANG Lisheng, MAO Jianjun. Identification and Expression Characteristics of Vitellogenin Gene in Chrysopa pallens Males[J]. Chinese Journal of Biological Control, 2021, 37(1): 77-85.

参考文献

[1] 赵敬钊. 对我国棉田草蛉发生规律的初步分析[J]. 武汉师范学院学报(自然科学版), 1981, 34(2):27-35.
[2] 胡坚. 大草蛉生活史及其在烟田的消长规律[J]. 烟草科技, 2012(2):80-82.
[3] 张帆, 王素琴, 罗晨, 等. 几种人工饲料及繁殖技术对大草蛉生长发育的影响[J]. 植物保护, 2004, 30(5):36-40.
[4] Slessor K N, Winston M, Conte L Y. Pheromone communication in the honeybee (Apis mellifera)[J]. Journal of Chemical Ecology, 2005, 31(11):2731-2745.
[5] 任炳忠, 赵卓. 松嫩平原蝗虫群落动态变化初步研究[J]. 吉林农业大学学报, 2000, 22(4):123-128.
[6] Guidugli K R, Piulachs M D, Bellés X, et al. Vitellogenin expression in queen ovaries and in larvae of both sexes of Apis mellifera[J]. Archives of Insect Biochemistry and Physiology, 2005, 59(4):211-218.
[7] Piulachs M D, Guidugli K R, Barchuk A R, et al. The vitellogenin of the honey bee, Apis mellifera:structural analysis of the cDNA and expression studies[J]. Insect Biochemistry and Molecular Biology, 2003, 33(4):459-465.
[8] Tufail Muhammad,Takeda Makio. Vitellogenin of the cockroach, Leucophaea maderae:nucleotide sequence, structure and analysis of processing in the fat body and oocytes[J]. Insect Biochemistry and Molecular Biology, 2003, 32(11):1469-1476.
[9] 刘昌燕. 大草蛉卵黄原蛋白基因功能研究[D]. 北京:中国农业科学院, 2013.
[10] 吴亚, 金翠霞. 草甸昆虫群落及其空间与时间结构[J]. 昆虫学报, 1980, 23(2):156-165.
[11] Livak K J, Schmittgen T D. Analysis of relative gene expression data using real-time quantitative PCR and the 2^-ΔΔCT method[J]. Methods, 2001, 25(4):402-408.
[12] Liu C, Mao J, Zeng F. Chrysopa septempunctata (Neuroptera:Chrysopidae) Vitellogenin functions through effects on egg production and hatching[J]. Journal of Economic Entomology, 2015, 108(6):2779-2788.
[13] 陶淑霞, 张帆, 李成德. 大草蛉卵黄发生的动态分析[J]. 吉林农业大学学报, 2004, 26(3):260-262.
[14] 奚剑飞. 椭圆食粉螨卵黄原蛋白基因对重金属铅胁迫的响应机制[D]. 南昌:南昌大学, 2019.
[15] 李志勇. 东方蜜蜂卵黄原蛋白基因转录模式[C]//中国农科院蜜蜂研究所. 国家蜂产业技术体系蜜蜂育种与授粉功能研究室学术研讨会暨中国养蜂学会蜜蜂育种专业委员会第四届第一次会议暨中国养蜂学会蜜源与蜜蜂授粉专业委员会第五届第一次会议论文汇编. 中国农科院蜜蜂研究所:中国养蜂学会, 2012, 4-17.
[16] 刘朝良, 王磊, 梶浦善太, 等. 天蚕卵黄原蛋白cDNA的克隆及序列分析[J]. 激光生物学报, 2006, 15(1):39-45.
[17] 王加伟,彭露, 邹明民, 等. 昆虫卵黄原蛋白受体(VgRs)及其主要功能综述[J]. 环境昆虫学报, 2016, 38(4):831-842.
[18] Tufail M, Lee J M, Hatakeyama M, et al. Cloning of vitellogenin cDNA of the American cockroach, Periplaneta americana (Dictyoptera), and its structural and expression analyses[J]. Archives of Insect Biochemistry and Physiology, 2000, 45(1):37-46.
[19] Tufail M, Hatakeyama M, Takeda M. Molecular evidence for two vitellogenin genes and processing of vitellogenins in the American cockroach, Periplaneta Americana[J]. Archives of Insect Biochemistry and Physiology, 2001, 48(2):72-80.
[20] Lee J M, Nishimori Y, Hatakeyama M, et al. Vitellogenin of the cicada Graptopsaltria nigrofuscata (Homoptera):analysis of its primary structure[J]. Insect Biochemistry and Molecular Biology, 2000, 30(1):1-7.
[21] 刘丽平. 烟盲蝽卵黄原蛋白基因表达的营养调控[C]//中国植物保护学会. 中国植物保护学会成立50周年庆祝大会暨2012年学术年会论文集. 中国植物保护学会:中国植物保护学会, 2012:574.
[22] Guidugli K R, Piulachs M D, Bellés X, et al. Vitellogenin expression in queen ovaries and in larvae of both sexes of Apis mellifera[J]. Archives of Insect Biochemistry and Physiology, 2005, 59(4):211-218.
[23] Moriyama M, Hosokawa T, Tanahashi M, et al. Suppression of bedbug's reproduction by RNA interference of vitellogenin[J]. PLoS ONE, 2016, 11(4):e0153984.
[24] Coelho R R, Firmino A A P, Macedo L L P D, et al. Vitellogenin knockdown strongly affects cotton boll weevil egg viability but not the number of eggs laid by females[J]. Meta Gene, 2016, 9(C):173-180.
[25] 严盈, 彭露, 万方浩. 昆虫卵黄原蛋白功能多效性:以蜜蜂为例[J]. 昆虫学报, 2010, 53(3):335-348.
[26] Engels W. Occurrence and significance of vitellogenins in female castes of social Hymenoptera[J]. American Zoologist, 1974, 14(4):1229-1237.
[27] Fluri P, Wille H, Gerig L, et al. Juvenile hormone,vitellogenin and hemocyte composition in winter worker honeybees (Apis mellifera)[J]. Experientia, 1977, 33(9):1240-1241.
[28] Amdam G V, Aase A L, Seehuus S C, et al. Social reversal of immunosenescence in honey bee workers[J]. Experimental Gerontology, 2005, 40(12):939-47.
[29] Seehuus S C, Norberg K, Gimsa U, et al. Reproductive protein protects functionally sterile honey bee workers from oxidative stress[J]. Proceedings of the National Academy of Sciences of the United States of America, 2006, 103(4):962-967.