【作者】 冯仁军；

【导师】 张银东； 金志强；

【作者基本信息】 华南热带农业大学 ， 作物遗传育种， 2004， 硕士

【摘要】 本研究利用96孔板和一对特异的引物，逐级从cDNA文库中筛选目标克隆，最终获得目标cDNA片段。与同位素杂交筛选法相比，该方法具有快速、简单、经济等优点，且无需使用同位素。本研究就是利用这一方法从香蕉cDNA文库中筛选出二条cDNA序列，其中一条序列是Mafra1，另一条序列是Maasr1。 在NCBI上的BLASTn和BLASTx比对结果表明Mafra1的功能尚不清楚，可能为一个新的基因；保守结构域分析结果表明Mafra1可能含有一个Exonuclease保守结构域；用OMIGA软件分析表明该片断包含了一个完整的ORF；用PROPSEARCH在线分析的结果表明Mafra1的编码产物可能与人的骨形态发生蛋白7前体(BMP-7)、分泌型糖蛋白前体(SGP)、外壳蛋白F(GPF)等基因具有较高同源。用Maasr1作BLASTn比对发现它可能与水稻ABA和胁迫诱导蛋白(Asr1)的mRNA有86％的同源，与甘蔗干旱胁迫蛋白(SoDip22)的mRNA有84％的同源，与百合花A23基因的mRNA有86％的同源，与野黑杏脱落胁迫成熟蛋白的mRNA有82％的同源；并且用GENSCAN_predicted进行在线分析，结果表明Maasr1包含了一个完整的ORF。 Southern杂交结果表明Maasr1在香蕉基因组中为双拷贝基因。Northern杂交结果表明：在采后香蕉果实中，随着时间的往后推移，Maasr1的表达量逐渐升高，这一变化与跃变以前的乙烯变化、果实软化的进程一致，所以Maasr1可能与香蕉果实的成熟相关，并且在果实成熟软化过程中可能是一个上调基因。更多还原

【Abstract】 The banana fruit cDNA library was subdivided into 96-well plate and propagated. The well carrying target cDNA sequence was determined by PCR using a pair of specific oligonucleotide primers in the research.This method was faster.more simple and inexpensive than the filter isotope hybridization technique,furthermore,it did not need using isotope.Two cDNA sequences had been isolated by this method in the banana fruit cDNA library :the one was Mafral,the other was Maasrl.The result of BLASTn and BLASTx showed that Mafral’s function was unknown,and might be a new gene.The result of conservative sequence analysis indicated that Mafral might contain a conservative domain of Exonuclease gene.The analyzing result of PROPSEARCH online made it known that Mafral might code a product which was highly homologous with Bone morphogenetic protein 7 precursor (BMP-7),Small/secreted glycoprotein precursor (SGP),and Capsid protein (F protein) (GPF).The analyzing result of OMIGA software made it clear that Mafral had an integrated open reading frame.The result of BLASTx showed that Maasrl might be homologous with Oryza sativa abscisic acid-inducible and stress-inducible protein (Asrl) mRNA (86%),Saccharum officinarum SoDip22 mRNA for drought inducible 22 kD protein ($4%)JLilium longiflorum anth (A23) mRNA (86%) , and Prunus armeniaca abscisic stress ripening protein homolog mRNA(82%). Maasrl included an integrated open reading frame by the analysis of GENSCAN_predicted online .Southern blot result showed that Maasrl had two copies in banana genome.Northern blot result indicated that the expression of Maasrl gradually ascended with time going on in harvested banana fruit.This change was consistent with the variational course of ethylene before climacteric period and fruit intenerating.The gene might associate with banana fruit ripening and might be an up-regulated gene in the fruit ripening and intenerating process.更多还原