申艳红;陈晓静;戴志林;

• 1:福建农林大学园艺学院·园艺植物遗传育种研究所

摘要(Abstract)：

【目的】获得诱导型基因Cp PGIP2启动子,并分析该启动子的功能。【方法】以番木瓜Cp PGIP2基因c DNA序列(HQ660394)搜索番木瓜基因组DNA序列,获得一段约2 000 bp的5’端上游DNA序列(ABIM01005077),参照该序列设计PCR特异引物,以番木瓜叶片DNA为模板克隆该启动子,并进行生物信息学分析。将3个启动子片段替换p CAMBIA1301载体中的Ca MV 35S启动子,构建缺失启动子表达载体,以GUS瞬时表达检测缺失启动子表达活性。【结果】获得了Cp PGIP2起始密码子上游长为1 981 bp的调控序列,经分析该序列含有真菌、脱落酸、赤霉素、光照等多种响应元件,为诱导型启动子。构建了1个启动子全长表达载体和2个5’端缺失启动子表达载体,分别命名为p D0-1981、p D1-1204和p D2-261。启动子在番木瓜果肉中的瞬时表达显示,长度为1 204 bp的启动子表达活性最强。并且,该启动子在根、茎、叶、果肉和愈伤组织中均有不同程度的表达,在近外果皮的果肉处和根部表达最明显。【结论】本研究获得了Cp PGIP2启动子序列,确定了表达活性最强的启动子长度,初步分析了启动子的表达模式和顺式作用元件,为进一步深入研究Cp PGIP2基因功能以及将该启动子应用于植物基因工程育种奠定了基础。

关键词(KeyWords)： 番木瓜;多聚半乳糖醛酸酶抑制蛋白;启动子;调控元件;载体构建

Abstract:

Keywords:

基金项目(Foundation): 福建农林大学园艺学院青年学术骨干培养基金(FAFU2012YYPY05);; 福建省自然科学基金资助项目(2010J05048)

作者(Author): 申艳红;陈晓静;戴志林;

Email:

DOI: 10.13925/j.cnki.gsxb.20150180

参考文献(References)：

• [1]CERVONE F,HAHN M G,DE LORENZO G,DARVILL A,ALBERSHEIM P.Host-pathogen interactions:XXXIII.A plantprotein converts a fungal pathogenesis factor into an elicitor ofplant defense responses[J].Plant Physiology,1989,90(2):542-548.
• [2]DEVLIN W S,GUSTINE D L.Involvement of the oxidative burstin phytoalexin accumulation and the hypersensitive reaction[J].Plant Physiology,1992,100(3):1189-1195.
• [3]TURNER J G,HOFFMAN R M.Effect of the polygalacturonaseinhibitor from pea on the hydrolysis of pea cell walls by the endo-polygalacturonase from Ascochyta pisi[J].Plant Pathology,1985,34(1):54-60.
• [4]DEVOTO A,LECKIE F,LUPOTTO E,CERVONE F,DE LOREN-ZO G.The promoter of a gene encoding a polygalacturonase-inhib-iting protein of Phaseolus vulgaris L.is activated by wounding butnot by elicitors or pathogen infection[J].Planta,1998,205(2):165-174.
• [5]LIANG F S,ZHANG K C,ZHOU C J,KONG F N,LI J,WANGB.Cloning,characterization and expression of the gene encodingpolygalacturonase-inhibiting proteins(PGIPs)of peach[Prunus persica(L.)Batch][J].Plant Science,2005,168:481-486.
• [6]尹涛,张上隆,刘敬梅,陈大明.提高外源基因在转基因植物中表达效率的途径[J].农业生物技术学报,2005,13(6):808-814.YIN Tao,ZHANG Shanglong,LIU Jingmei,CHEN Daming.Meth-ods of improving heterogeneous gene expression level in transgen-ic plants[J].Journal of Agricultural Biotechnology,2005,13(6):808-814.
• [7]PAUL W,HODGE R,SMARTT S,DRAPER J,SCOTT R.Theisolation and characterisation of the tapetum-specific Arabidopsis thaliana A9 gene[J].Plant Molecular Biology,1992,19(4):611-622.
• [8]柏亚男,吴茂森,何晨阳.水稻基因启动子Os BTF3p的克隆和启动活性分析[J].中国农业科学,2009,42(3):862-868.BAI Yanan,WU Maosen,HE Chenyang.Molecular cloning andactivity analysis of a rice gene promoter Os BTF3p[J].Scientia Agri-cultura Sinica,2009,42(3):862-868.
• [9]毛娟,陆璐,陈佰鸿,禇明宇,赵长增.甜瓜Cm ACOⅠ启动子组织特异性表达研究[J].园艺学报,2013,40(6):1101-1109.MAO Juan,LU Lu,CHEN Baihong,CHU Mingyu,ZHAO Chang-zeng.Studies on tissue specific regulation of Cm ACOⅠpromoterin melon[J].Acta Horticulturae Sinica,2013,40(6):1101-1109.
• [10]李广平,张长青,章镇,曹福亮.中国李pgip启动子的克隆及调控元件分析[J].园艺学报,2009,36(10):1425-1430.LI Guangping,ZHANG Changqing,ZHANG Zhen,CAO Fu-liang.Cloning of pgip promoter from Prunus salicina and the com-putational identification of its regulatory element[J].Acta Horticul-turae Sinica,2009,36(10):1425-1430.
• [11]李广平,张长青,章镇.梅PGIP基因的启动子克隆及生物信息学分析[J].西北植物学报,2010,30(5):883-887.LI Guangping,ZHANG Changqing,ZHANG Zhen.Cloning andsequence analyzing of PGIP promoter from Prunus mume[J].ActaBotanica Boreali-Occidentalia Sinica,2010,30(5):883-887.
• [12]王秀云,张计育,古咸彬,高志红,章镇,俞明亮,张妤艳.桃Pp PGIP1启动子的分离与功能分析[J].园艺学报,2012,39(7):1263-1270.WANG Xiuyun,ZHANG Jiyu,GU Xianbin,GAO Zhihong,ZHANG Zhen,YU Mingliang,ZHANG Yuyan.Isolation and func-tional analysis of Pp PGIP1 promoter in peach[J].Acta Horticul-turae Sinica,2012,39(7):1263-1270.
• [13]JOUBERT D A,DE LORENZO G,VIVIER M A.Regulation ofthe grapevine polygalacturonase-inhibiting protein encoding gene:expression pattern,induction profile and promoter analysis[J].Journal of Plant Research,2013,126(2):267-281.
• [14]申艳红,陈晓静,李荣荣,卢秉国,何玮毅.2个番木瓜多聚半乳糖醛酸酶抑制蛋白基因的克隆与分析[J].热带作物学报,2011,32(5):861-865.SHEN Yanhong,CHEN Xiaojing,LI Rongrong,LU Bingguo,HE Weiyi.Cloning and analysis of two polygalacturonase inhibit-ing protein genes from papaya[J].Chinese Journal of TropicalCrops,2011,32(5):861-865.
• [15]MING R,HOU S B,FENG Y,YU Q,DIONNE-LAPORTE A.The draft genome of the transgenic tropical fruit tree papaya(Carica papaya Linnaeus)[J].Nature,2008,452:991-996.
• [16]王莹,韩烈保,曾会明.高等植物启动子克隆方法的研究进展[J].生物技术通报,2007(3):97-100.WANG Ying,HAN Liebao,ZENG Huiming.Advances on stud-ies of plant promoters[J].Biotechnology Bulletin,2007(3):97-100.
• [17]ZHANG F,WANG Z,HUANG Q S,HUANG L P,YANG Q.Cloning and characterization of a PGIP-encoding gene from Solanum torvum[J].Agricultural Sciences in China,2010,9(6):921-927.
• [18]HU D,DAI R,WANG Y,ZHANG Y,LIU Z,FANG R,ZHAOW,LI L,LIN Q,LI L.Molecular cloning,sequence analysis,and expression of the polygalacturonase-inhibiting protein(PGIP)gene in mulberry[J].Plant Molecular Biology Reporter,2012,30(1):176-186.
• [19]De LORENZO G,DOVIDIO R,CERVONE F.The role of polyga-lacturonase-inhibiting proteins(PGIPs)in defense against patho-genic fungi[J].Annual Review of Phytopathology,2001,39(1):313-335.