基于EST数据库的葡萄APETALA2基因cDNA克隆及其表达分析Cloning and expression analysis of APETALA2 gene from grapevine (Vitis vinifera)based on EST database
王晨;刘洪;房经贵;宋长年;曹雪;杨光;章镇;
摘要(Abstract):
APETALA2(AP2)是拟南芥生长发育特别是花器官发育过程中重要的调控基因。利用生物信息学方法以拟南芥APETALA2基因cDNA序列作为模板,对葡萄EST数据库进行同源检索筛选,电子克隆出葡萄AP2基因(Vv-AP2)cDNA序列。以香悦葡萄花cDNA为模板,根据电子克隆的葡萄AP2基因cDNA序列设计特异引物,分别利用RACE技术和特异PCR技术获得该基因3’末端和5’末端,序列拼接后获得葡萄的APETALA2基因的cDNA全长。该cDNA的全长为2 208 bp,命名为Vv-AP2。Vv-AP2核苷酸序列有一个1 536 bp完整的开放读码框(ORF),其5’与3’末端非翻译区分别为268 bp和376 bp。其中,3’末端含有28 bp的Ploy+(A)。该基因在GenBank基因数据库的注册号为FJ809943。氨基酸推导结果表明该cDNA共编码了511个氨基酸,具备完全保守的核定位信号序列(KKSR)以及2个高度保守的重复序列即AP2结构域。分别采用半定量RT-PCR和SYBR Green I实时定量RT-PCR初步分析了Vv-AP2在葡萄叶、茎、花序、花等不同器官中的表达水平。其中,Vv-AP2在花序和花中的表达量明显高于叶和茎。
关键词(KeyWords): 葡萄;Vv-AP2;基因克隆;表达分析
基金项目(Foundation): 国家博士后基金(20080440161)
作者(Author): 王晨;刘洪;房经贵;宋长年;曹雪;杨光;章镇;
Email:
DOI: 10.13925/j.cnki.gsxb.2010.02.009
参考文献(References):
- [1]JOFUKU K D,BOER BGWD,MONAGU M V,OKAMURO J K.Control of arabidopsis flower and seed development by the homeoticgene APETALA2[J].Plant Cell,1994,6(9):1211-1225.
- [2]VIJAYRAGHAVAN U,PRASAD K,MEYEROWITZ E.Specificationand maintenance of the floral meristem:interactions betweenpositively-acting promoters of flowering and negative regulators[J].Current Science,2005,89(11):1835-1843.
- [3]SHANNON S,MEEKS-WAGNER D R.Genetic interactions thatregulate inflorescence development in arabidopsis[J].Plant Cell,1993,5:639-655.
- [4]CHEN X.A MicroRNA as a translational repressor of APETALA2 inarabidopsis flower development[J].Science,2004,303:2022-2025.
- [5]JOFUKU K D,OMIDYAR P K,GEE Z,OKAMURO J K.Control ofseed mass and seed yield by the floral homeotic gene APETALA2[J].Proc Natl Acad Sci USA,2005,102(8):3117-3122.
- [6]OHTO M,FISCHER R,GOLDBERG R B,NAKAMURA K,HARA-DA J J.Control of seed mass by APETALA2[J].Proc Natl Acad SciUSA,2005,102(8):3123-3128.
- [7]W譈RCHUM T,GROβ-HARDT R,LAUX T.APETALA2 regulatesthe stem cell niche in the arabidopsis shoot meristem[J].Plant Cell,2006,18:295-307.
- [8]LAUTER N,KAMPANI A,CARLSON S,GOEBEL M,MOOSE SP.microRNA172 down-regulates glossy15 to promote vegetativephase change in maize[J].Proc Natl Acad Sci USA,2005,102(26):9412-9417.
- [9]ZHOU Sheng-mei,SU Hong-yan,WANG Lei,ZHANG Xian-sheng,SHU Huai-rui.Cloning and transformation of apetala2 homologuesgenes from apple[J].Acta Horticulturae Sinica,2006,33(2):239-243.周盛梅,宿红艳,王磊,张宪省,束怀瑞.苹果apetala2同源基因的克隆和转化研究[J].园艺学报,2006,33(2):239-243.
- [10]SU Hong-yan,ZHOU Sheng-mei,WANG Lei,ZHANG Xian-sheng,SHU Huai-rui.Cloning and expression analysis of APETALA2 ho-mologous genes in strawberry[J].Acta Botanica Boreali-OccidentaliaSinica,2005,25(10):1937-1942.宿红艳,周盛梅,王磊,张宪省,束怀瑞.草莓APETALA2同源基因的克隆及表达分析[J].西北植物学报,2005,25(10):1937-1942.
- [11]ZHAO Qi,WANG Tai,WEI Xiao-di.Function of AP2 gene duringflor al organs development in higher plant[J].Chinese Journal of Trop-ical Agriculture,2005,25(3):50-56.赵奇,王台,魏小弟.AP2基因在高等植物花器官发育过程中的作用[J].热带农业科学,2005,25(3):50-56.
- [12]MAES T,VAN DE STEENE N,ZETHOF J,KARIMI M,D'HAUWM,MARES G,VAN MONTAGU M,GERATS T.Petunia AP2-likegenes and their role in flower and seed development[J].Plant Cell,2001,13(2):229-244.
- [13]MLOTSHWA S,YANG Z,KIM Y,CHEN X.Floral patterning de-fects induced by arabidopsis APETALA2 and microRNA172 expres-sion in nicotiana benthamiana[J].Plant Mol Biol,2006,61:781-793.
- [14]JAILLON O,AURY J-M,NOEL B.The grapevine genome sequencesuggests ancestral hexaploidization in major angiosperm phyla[J].Nature,2007,449:463-467.
- [15]FANG Jing-gui,LIU Hong,YANG Guang.The star grape winecultivar in genomic sequencing era―Pinot Noir[J].Sino-overseasGrapevine&Wine,2008(3):57-59.房经贵,刘洪,杨光.基因组学研究时代的葡萄酿造明星—黑比诺[J].中外葡萄与葡萄酒,2008(3):57-59.
- [16]BOSS P K,THOMAS M R.Tendrils,inflorescences and fruitfulness:a molecular perspective[J].Australian Journal of Grape and WineResearch,2000,6:168-174.
- [17]RAMAKERS C,RUIJTERA J M,LEKANNE DEPREZA R H,MOORMAN A F M.Assumption-free analysis of quantitative real-time polymerase chain reaction(PCR)data[J].Neuroscience Letters,2003,339:62-66.
- [18]KIM S,SOLTIS P S,WALL K,SOLTIS D E.Phylogeny and domainevolution in the APETALA2-like gene family[J].Mol Biol Evol,2005,23:107-120.
- [19]QIN Jie,WANG Wu,ZUO Kai-jing.Origin of AP2 gene family andcotton AP2 transcription factors′roles in plant resistance[J].CottonScience,2005,17(6):366-370.秦捷,王武,左开井.AP2基因家族的起源和棉花抗病转录因子在抗病中的的作用[J].棉花学报,2005,17(6):366-370.