阳永学;程维舜;曾红霞;张娜;施先锋;孙玉宏;

• 1:武汉市农业科学研究所

摘要(Abstract)：

[目的]克隆西瓜半胱氨酸蛋白酶基因ClCP1,并对其进行生物信息学分析。[方法]以西瓜花蕾cDNA为模板,借助西瓜全基因组数据库,采用RT-PCR方法对西瓜半胱氨酸蛋白酶基因进行克隆并进行生物信息学分析。[结果]克隆得到一个开放阅读框长度为960 bp的序列,命名为ClCP1。该基因编码319个氨基酸。ClCP1基因的保守结构域分析结果表明,ClCP1属于木瓜蛋白酶家族基因,与已知其他植物半胱氨酸蛋白酶在氨基酸序列上有56%~85%的相似性。聚类分析结果显示,ClCP1与同为葫芦科的黄瓜CP基因亲缘关系较近。[结论]ClCP1属于西瓜木瓜蛋白酶家族新基因,为进一步研究西瓜半胱氨酸蛋白酶基因ClCP1的表达和功能奠定了基础。

关键词(KeyWords)： 西瓜;ClCP1;基因克隆;生物信息学

Abstract:

Keywords:

基金项目(Foundation): 武汉市农业科学技术研究院创新项目(CX201242)

作者(Author): 阳永学;程维舜;曾红霞;张娜;施先锋;孙玉宏;

Email:

DOI: 10.13989/j.cnki.0517-6611.2013.28.094

参考文献(References)：

• [1]LEE S,JUNG K H,AN G,et al.Isolation and characterization of a rice cysteine protease gene,OsCP1,using T-DNA gene trap system[J].Plant Molecular Biology,2004,154:755-765.
• [2]ZHANG X M,WANG Y,L X M,et al.NtCP56,a new cysteine protease in Nicotiana tabacum L,involved in pollen grain development[J].Journal of Experimental Botany,2009,60:1569-1577.
• [3]GUAN H C,LIN T H,MEE N Y,et al.Molecular characterization of a senescence-associated gene encoding cysteine proteinase and its gene expression during leaf senescence in sweet tomato[J].Plant and Cell Physiology,2002,43(9):984-991.
• [4]TRIPATHI S K,SINGH A P,ANIRUDDHA P,et al.Transcriptional activation of a 37 kDa ethylene responsive cysteine protease gene,RbCP1,is associated with protein degradation during petal abscission in rose[J].Journal of Experimental Botany,2009,60(7):2035-2044.
• [5]NOH Y S,AMASINO R M.Regulation of developmental senescence is conserved between Arabidopsis and Brassica napus[J].Plant Mololecular Biology,?1999,41(2):195-206.
• [6]GUO S G,ZHANG J G,SUN H H,et al.The draft genome of watermelon(Citrullus lanatus)and resequencing of 20 diverse accessions[J].Nature Genetics,2013,45:51-58.
• [7]GUO S G,LIU J A,ZHENG Y,et al.Characterization of transcriptome dynamics during watermelon fruit development:sequencing,assembly,annotation and gene expression profiles[J].BMC Genomics,2011,12:454-467.
• [8]LIU N,YANG J H,GUO S G,et al.Genome-wide identification and comparative analysis of conserved and novel microRNAs in grafted watermelon by high-throughput sequencing[J].Plos ONE,2013,8(2):57359.
• [9]陈惠明,卢向阳,许亮,等.黄瓜、西瓜和南瓜EIN3基因片段的克隆与序列分析[J].农业生物技术学报,2007,15(4):668-672.
• [10]曹迪,许勇,郭绍贵,等.西瓜和黄瓜乙烯受体ETRI基因片段的克隆与序列比较分析[J].华北农学报,2009,24(5):59-63.
• [11]康保珊,赵文恩.西瓜八氢番茄红素合成酶基因片段的克隆和序列分析[J].西北植物学报,2007,27(5):859-863.
• [12]张建新,刘起丽,吴云锋.西瓜花叶病毒外壳蛋白基因的克隆与原核表达[J].河南师范大学学报:自然科学版,2008,36(4):117-123.
• [13]李严曼,朱磊,杨景华,等.西瓜交替氧化酶AOX2基因的克隆与分析[J].果树学报,2011,28(5):909-914.
• [14]CHO S M,KANG E Y,MIN K H,et al.A positive regulatory role of the watermelon ClWRKY70 gene for disease resistance in transgenic Arabidopsis thaliana[J].Biologia Plantarum,2012,56(3):560-565.
• [15]张曼,羊杏平,王薇薇,等.西瓜ClPDF2.6基因的鉴定与表达谱分析[J].江苏农业学报,2013,29(4):831-836.
• [16]MARTI-RENOM M A,MADHUSUDHAN M S,FISER A,et al.Reliability of assessment of protein structure prediction methods[J].Structure,2002,10(1):435.
• [17]严秀蕊,张大生,梁婉琪,等.水稻半胱氨酸蛋白酶OsCP2的特征分析及其原核表达与纯化[J].上海交通大学学报:农业科学版,2010,28(2):141-146.
• [18]KARRER K M,PEIFFER S L,DITOMAS M E.Two distinct gene subfamilies within the family of cysteine protease genes[J].Proceedings of the National Academy of Sciences,1993,90:3063-3067.
• [19]KAMPHUIS I G,DRENTH J,BAKER E N.Thiol proteases:comparative studies based on the high-resolution structures of papain and actinidin,and on amino acid sequence information from cathepsins B and H,and stem bromelain[J].Journal of Molecular Biology,1985,182:317-329.
• [20]GUERRERO C,CALLE M,REID M S,et al.Analysis of the expression of two thioprotease genes from day lily(Hemerocallis spp.)during flower senescence[J].Plant Molecular Biology,1998,36:565-571.
• [21]BEYENE G,FOYER C H,KUNERT K J.Two new cysteine proteinases with specific expression patterns in mature and senescent tobacco(Nicotiana tabacum L.)leaves[J].Journal of Experimental Botany,2006,57:1431-1443.
• [22]KOIZUMI M,YAMAGUCHI-SHINOZAKI K,TSUJI H,et al.Structure and expression of two genes that encode distinct drought-inducible cysteine proteinases in Arabidopsis thaliana[J].Gene,1993,129:175-182.
• [23]MCLELLAN H,GILROY E M,YUN B W,et al.Functional redundancy in the Arabidopsis Cathepsin B gene family contributes to basal defence,the hypersensitive response and senescence[J].New Phytologist,2009,183:408-418.
• [24]SOLOMON M,BELENGHI B,DELLEDONNE M,et al.The involvement of cysteine proteases and protease inhibitor genes in the regulation of programmed cell death in plants[J].Plant Cell,1999,11:431-444.
• [25]LI N,ZHANG D S,LIU H S,et al.The rice tapetum degeneration retardation gene is required for tapetum degradation and anther development[J].Plant Cell,2006,18:2999-3014.
• [26]SUN Z X,ZHOU Y.Purification of Antifungal Protein from Biocontrol Strain XG-1 against Fusarium Wilt of Watermelon[J].Agricultural Science&Technology,2013,14(3):407-410.
• [27]胡宝刚,刘莉,焦定量.强雌性系西瓜ACC合成酶基因的克隆及其序列分析[J].华北农学报,2011(1):67-71.