2023 04 v.34;No.112 608-617
褐飞虱卵黄原蛋白基因NlVg1的克隆及表达分析
基金项目(Foundation):
国家自然科学基金项目(No.U21A20223,31672026,32011530117);;
浙江省科技计划项目(No.LY20C140005,2019C02015,2022C02047);;
中国计量大学科研标志性发展专项项目(No.2020YW27)
邮箱(Email):
haopy@cjlu.edu.cn;
DOI:
中文作者单位:
中国计量大学生命科学学院浙江省生物计量检验检疫技术重点实验室;瑞典农业科学大学乌普萨拉生物中心植物生物学系;
摘要(Abstract):
目的:卵黄原蛋白是昆虫卵黄磷蛋白的前体，也是卵母细胞成熟和胚胎发育阶段最为重要的生殖蛋白。本文研究褐飞虱Nilaparvata lugens St?l卵黄原蛋白基因的表达规律，旨在为筛选害虫防治靶标及敏感期提供线索。方法:根据褐飞虱转录组测序信息，对预测编码卵黄原蛋白的Unigene 40792序列(NlVg1)进行了克隆，应用实时荧光定量PCR技术对NlVg1在褐飞虱不同发育时期以及不同水稻品种上的表达规律进行了研究。结果:克隆的NlVg1开放阅读框长1 212 bp,编码的蛋白含403个氨基酸残基。通过对转录水平的表达分析发现，NlVg1在褐飞虱1～4龄若虫期不表达，从5龄开始有少量表达，在羽化3 d的成虫时期表达量最高；在抗性水稻品种的表达量低于感性品种。结论:本研究克隆了褐飞虱的1个卵黄原蛋白相关基因NlVg1的全长cDNA开放阅读框，对其编码的氨基酸序列进行了生物信息学分析，并通过荧光定量PCR技术对褐飞虱在不同发育时期以及不同水稻品种饲养条件下的该基因的表达规律进行研究。研究结果为后期选择合适的时期对目的基因进行RNAi等功能研究提供了线索。
关键词(KeyWords):
褐飞虱;;卵黄蛋白;;基因克隆;;表达规律;;实时荧光定量PCR
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参考文献
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[23] 赵焕英，包金风.实时荧光定量PCR技术的原理及其应用研究进展[J].中国组织化学与细胞化学杂志，2007,16(4):492-497.ZHAO H Y,BAO J F.The principle and application research progress of real-time fluorescence quantitative PCR technology[J].Chinese Journal of Histochem Istry and Cytochem Istry,2007,16(4):492-497.
[24] 王光华，赵伟春，程家安.实时荧光定量PCR技术及其在昆虫学研究中的应用[J].昆虫学报，2008,51(12):1293-1303.WANG G H,ZHAO W C,CHENG J A.Real-time fluorescent quantitative PCR technique ang its applications in entomological studies[J].Acta Entomologica Sinica,2008,51(12):1293-1303.
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[33] 李洁.抗虫水稻对褐飞虱抗性研究及褐飞虱apterousA基因的功能研究[D].武汉：华中农业大学，2013:1-41.LI J.Study on the Resistance of Insect Resistant Rice to Brown Planthopper and the Function of the Brown Planthopper adaptousA Gene[D].Wuhan:Huazhong Agricultural University,2013:1-41.
[34] ENGELBERTH J,ALBORN H T,SCHMELZ E A,et al.Airborne signals prime plants against insect herbivore attack[J].Proceedings of the National Academy of Sciences of the United States of America,2004,101(6):1781-1785.
[35] 凌冰，董红霞，张茂新，等.水稻麦黄酮对褐飞虱的抗性潜力[J].生态学报，2007(4):1300-1307.LING B,DONG H X,ZHANG M X,et al.Potential resistance of tricin in rice against brown planthopper Nilaparvata lugens( St?l)[J].Acta Ecologica Sinica,2007(4):1300-1307.
[2] CAO T T,Lü J,LOU Y G,et al.Feeding-induced interactions between two rice planthoppers,Nilaparvata lugens and Sogatella furcifera(Hemiptera:delphacidae):Effects on feeding and honeydew excretion[J].Environmental Entomology,2013,42(6):1281-1291.
[3] BACKUS E A,SERRANO M S,RANGER C M.Mechanisms of hopperburn:An overview of insect taxonomy,behavior,and physiology[J].Annual Review of Entomology,2005,50:125-151.
[4] CHEN J W,WANG L,PANG X F,et al.Genetic analysis and fine mapping of a rice brown planthopper(Nilaparvata lugens St?l) resistance gene bph19(t)[J].Molecular Genetics and Genomics,2006,276(4):1617-4615.
[5] JENA K K,JEUNG J U,LEE J H,et al.High-reso lution mapping of a new brown planthopper(BPH) resistance gene,Bph18(t),and marker-assisted selection for BPH resistance in rice(Oryza sativa L.)[J].Theoretical and Applied Genetics,2006,112:288-297.
[6] 蔡文霞.水稻褐飞虱的发生及防治[J].安徽农学通报，2008,14(16):67.CAI W X.The occurrence and control of brown planthopper[J].Anhui Agricultural Science Bulletin,2008,14(16):67.
[7] IKEDA R,KANEDA C.Trisomic analysis of the gene Bhp1 for resistance to the brown planthopper(Nilaparavata lugens St?l) in rice[J].Japanese Journal of Breeding,1983,33(1):40-44.
[8] 刘宇锋，李容柏，杨朗，等.水稻褐飞虱抗性基因研究进展[J].广西农业科学，2007,38(1):11-15.LIU Y F,LI R B,YANG L,et al.Advances in resistance gene of brown planthopper in rice[J].Guangxi Agricultural Sciences,2007,38(1):11-15.
[9] ZHANG Q F.Strategies for developing Green Super Rice[J].Proceedings of the National Academy of Sciences of the United States of America,2007,104:16402-16409.
[10] 吴昌军，姜恭好，李信，等.利用DH群体动态检测水稻抗褐飞虱数量性状基因位点[J].分子植物育种，2005(4):456-462.WU C J,JIANG G H,LI X,et al.Dynamic detection of quantitative trait loci for brown planthopper resistance in rice using DH population dynamics[J].Molecular Plant Breeding Mol Plant Breed,2005(4):456-462.
[11] TANAKA K.Quantitative genetic analysis of biotypes of the brown planthopper Nilaparvata lugens:Heritability of virulence to resistant rice varieties[J].Entomologia Experimentalis Et Applicata,1999,90:279-287.
[12] 吕仲贤，俞晓平，郑许松，等.褐飞虱致害性变异过程及其体内酶的变化[J].昆虫学报，1997,40(增刊):122-127.LV Z X,YU X P,ZHENNG X S,et al.The process of virulence variation and changes in enzymes in the body of brown planthopper[J].Acta Entomologica Sinica,1997,40(suppl):122-127.
[13] CHEN J,ZHANG D,YAO Q,et al.Feeding-based RNA interference of a trehalose phosphate synthase gene in the brown planthopper,Nilaparvata lugens[J].Insect Molecular Biology,19(6):777-786.
[14] U TARABHAND P,BUNLIPATANON P.Plasma vitellogenin of grouper(Epinephelus malabaricus):isolation and properties[J].Comp Biochem Physiol,1996,115C:101-110.
[15] PAN M L,BELL W J,TELFER W H.Vitellogenic blood protein syntheis by insect fat body[J].Science,1969,165:393-394.
[16] 周庆祥，江桂斌.卵黄蛋白原的分离定及其在环境内分泌干扰物质筛选中的应用[J].化学进展，2003,15(1):67-73.ZHOU Q X,JIANG G B.Separation and determination of vitellogenin and its application as a biomarker for the screening of environmental endocrine disrupting chemicals[J].Progress in Chemistry,2003,15(1):67-73.
[17] WALLACE R A.The Vertebrate Ovary:Comparative Biology and evolution[M].New York,Plenum Press,1978,469-502.
[18] TABORSKY G.Iron binging by phosvitin and its conformational consequences[J].Biological Chemistry,1980,255:2976-2985.
[19] 张年国，张颖，孙大江，等.卵黄蛋白原的发生、结构及功能研究现状[J].水产学杂志，2007,20(1):10.ZHANG N G,ZHANG Y,SUN D J,et al.Current status of research on the occurrence,structure,and function of vitellinogen[J].Chinese Journal of Fisheries,2007,20(1):10.
[20] SHARROCK W J,ROSENWASSER T A,GOUL J ,et al.Sequence of lamprey vitellogenin:Implications for the lipovitellin crySt?l structure[J].Molecular Biology,1992,226(3):903-907.
[21] 戴华国，衣维贤.褐飞虱卵黄蛋白的分离及其生化特性[J].昆虫学报，2006,49(1):29-33.DAI H G,YI W X.Separation and characterization of vitellin of Nilaparvata lugens(St?l)(Hemiptera:delphacidae)[J].Acta Entomologica Sinica,2006,49(1):29-33.
[22] 马月萍，戴思兰，马艳蓉.荧光定量PCR技术在植物研究中的应用[J].生物技术通报，2011,7:37-44.MA Y P,DAI S L,MA Y R.Application of technique of quantitative real-time PCR in research of plants[J].Biotechnology Bulletin,2011,7:37-44.
[23] 赵焕英，包金风.实时荧光定量PCR技术的原理及其应用研究进展[J].中国组织化学与细胞化学杂志，2007,16(4):492-497.ZHAO H Y,BAO J F.The principle and application research progress of real-time fluorescence quantitative PCR technology[J].Chinese Journal of Histochem Istry and Cytochem Istry,2007,16(4):492-497.
[24] 王光华，赵伟春，程家安.实时荧光定量PCR技术及其在昆虫学研究中的应用[J].昆虫学报，2008,51(12):1293-1303.WANG G H,ZHAO W C,CHENG J A.Real-time fluorescent quantitative PCR technique ang its applications in entomological studies[J].Acta Entomologica Sinica,2008,51(12):1293-1303.
[25] 马艳.褐飞虱表皮蛋白基因NlICP克隆及功能研究[J].昆虫学报，2013,56(11):1244-1251.MA Y.Molecular cloning and function analysis of cuticular protein gene NlICP in the rice brown planthopper,Nilaparvata lugens (Hemiptera:Delphacidae)[J].Acta Entomologica Sinica,2013,56(11):1244-1251.
[26] 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.
[27] CAMEJO A,GOLD D A,LU D,et al.Identification of three novel Toxoplasma gondii rhoptry proteins[J].International Journal for Parasitology,2014,44(2):147-160.
[28] 葛圣雷，陈主初，肖志强，等.分泌性蛋白质的研究策略[J].生命的化学，2005(6):476-477.GE S L,CHEN Z C,XIAO Z Q,et al.Research strategies for secretory proteins[J].Chemistry of Life,2005(6):476-477.
[29] 姬金亮，张瑞娟，李雅彬，等.褐飞虱KCTD3基因克隆，序列分析及其时空表达研究[J].中国计量大学学报，2022,33(3):423-431.JI J L,ZHANG R J,LI Y B,et al.Study on cloning,sequence analysis and spatio-temporal expression of KCTD3 gene in Nilaparvata lugens( St?l)[J].Journal of China University of Metrology,2022,33(3):423-431.
[30] 张焓娇.Mudgo水稻多代饲养影响褐飞虱致害性变异的研究[D].杭州：浙江农林大学，2018:18-37.ZHANG H J.Virulence Variation of the Brown Planthopper(Nilaparvata lugens) on Multi-Generational Resistant Rice Variety Mudgo[D].Hangzhou:Zhejiang Agriculture & Forestry University,2018:18-37.
[31] 唐明远，罗泽民，彭忠魁.关于杂交水稻中r—氨基丁酸等氨基酸含量与抗褐飞虱的相关性[J].湖南农学院学报，1979(4):27-30.TANG M Y,LUO Z M,PENG Z K.The correlation between the content of amino acids such as r-aminobutyric acid and resistance to brown planthopper in hybrid rice[J].Journal of Hunan Agricultural University,1979(4):27-30.
[32] DADD R H.Nutrition:Organisms[M]//Regulation:Digestion,Nutrition,Excretion.Amsterdam:Elsevier,1985:313-390.
[33] 李洁.抗虫水稻对褐飞虱抗性研究及褐飞虱apterousA基因的功能研究[D].武汉：华中农业大学，2013:1-41.LI J.Study on the Resistance of Insect Resistant Rice to Brown Planthopper and the Function of the Brown Planthopper adaptousA Gene[D].Wuhan:Huazhong Agricultural University,2013:1-41.
[34] ENGELBERTH J,ALBORN H T,SCHMELZ E A,et al.Airborne signals prime plants against insect herbivore attack[J].Proceedings of the National Academy of Sciences of the United States of America,2004,101(6):1781-1785.
[35] 凌冰，董红霞，张茂新，等.水稻麦黄酮对褐飞虱的抗性潜力[J].生态学报，2007(4):1300-1307.LING B,DONG H X,ZHANG M X,et al.Potential resistance of tricin in rice against brown planthopper Nilaparvata lugens( St?l)[J].Acta Ecologica Sinica,2007(4):1300-1307.
基本信息:
DOI：
中图分类号:S435.112.3
引用信息:
[1]陈桐桐,张旖旎,郝培应等.褐飞虱卵黄原蛋白基因NlVg1的克隆及表达分析[J].中国计量大学学报,2023,34(04):608-617.
基金信息:
国家自然科学基金项目(No.U21A20223,31672026,32011530117);; 浙江省科技计划项目(No.LY20C140005,2019C02015,2022C02047);; 中国计量大学科研标志性发展专项项目(No.2020YW27)
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