【作者】 潘华清；

【导师】 何龙飞；

【作者基本信息】 广西大学 ， 作物栽培学与耕作学， 2008， 硕士

【摘要】 用异硫氰酸胍法、CTAB法和Trizol法等三种方法提取飞机草总RNA均取得较好效果,但异硫氰酸胍法最适合本实验使用。根据菊科植物紫茎泽兰P450CYP75基因(AY954290)和拟南芥CYP75家族成员的核酸序列同源区设计引物,用RT-PCR方法首次从飞机草、泽兰、佩兰中获得大小分别为467bp、467bp、470bp的cDNA序列,分别命名为:FJC.CYP.M;ZL.CYP.M;PL.CYP.M。同源性分析表明,3个序列同源性达99%,同时与AY954290具有90%的同源性,说明它们都来自细胞色素CYP75家族。根据FJC.CYP.M核酸序列设计特异性引物,通过RACE-PCR方法克隆飞机草P450基因全长。3’RACE-PCR得到309bp cDNA序列,PolyA尾巴出现,命名为FJC.CYP.3。5’RACE-PCR得到702bp cDNA序列,命名为FJC.CYP.5。经比对发现,它与紫茎泽兰CYP75基因全长还差364bp,因此继续从紫茎泽兰CYP75基因前端设计上游引物,从FJC.CYP.5核酸序列设计下游引物,进行PCR,克隆得到一条长为384bp的cDNA序列,命名为FJC.CYP.5e。把所有飞机草细胞色素p450片段测序结果进行拼接,得到长为1653bp的cDNA序列,命名为FJC.CYP.COM,推导编码509个氨基酸,开放阅读框为31bp-1560bp,出现了3个经典的细胞色素P450氨基酸保守区域,其中一个为F××G×R×C×G,起始密码子ATG前27bp处出现了启动子序列TATAbox。功能预测表明飞机草P450蛋白为一个亲水性稳定蛋白,有内外膜共9个跨膜螺旋,定位于线粒体中。根据泽兰、佩兰中间片段设计引物,进行3’RACE-PCR,分别得到两条长为340bp、625bp的cDNA片段,分别命名为ZL.CYP.3、PL.CYP.3。经比对,初步推测ZL.CYP.3片段基因属于P450家族里某个叶绿体束缚蛋白基因,其具体的功能还有待进一步研究;PL.CYP.3片段属于P450家族的赤霉素控制蛋白基因,初步估计它参与植物激素赤霉素的合成。更多还原

【Abstract】 The effect of guanidine isothiocyanate method,CTAB method,and Trizol method on total RNA extraction from Eupatorium adoratum were compared,three methods had good effect but the first one was the best.Three cDNA fragments were obtained from Eupatorium adoratum,Lycopuslucidus turcz,and Eupatorium fortunei by RT-PCR stragtegy using primers based on the homologus nucleotide sequences of CYP75(AY954290)in Eupatorium adenophorum and members of CYP75 in Arabidopsis thliana.One was 467bp from Eupatorium adoratum named FJC.CYP.M,the second was 467bp from Lycopuslucidus turcz named ZL.CYP.M,the third was 470bp from Eupatorium fortunei named PL.CYP.M.Three sequences shared a homology of 99%with each other and 90%with CYP75 in Eupatorium adenophorum (AY954290)and were demonstrated to come from p450 super familiy.Two cDNA nucleotide sequences were gotten from Eupatorium adoratum by RACE-PCR using primers designed from FJC.CYP.M. FJC.CYP.3 was 309bp with ployA end obtained by 3’RACE-PCR,and FJC.CYP.5 is 702bp obtained by 5’RACE-PCR.Compared with the 5’end of Eupatorium adenophorum CYP75 gene,FJC.CYP.5 lacked 364bp.A 384bp cDNA fragments named FJC.CYP.5e was obtained by RT-PCR using primers designed from FJC.CYP.5 and the 5’end of CYP75 gene in Eupatorium adenophorum.One full-length cDNA nucleotide sequence named FJC.CYP.COM was gotten by combining all the fragments above in Eupatorium adoratum.It had 1653bp encoding 509 amino acids and had a ORF from 31bp-1560bp.The sequence included three conserved regions of P450 such as F××G×R×C×G, and a promotor sequence TATA box could be found in 27bp before the transcriptional initiation site of ATG.FJC.CYP.COM is a stable, hydropathicity protein with 9 transmembrate helices,which were located in mitochondria.Two cDNA fragments were obtained by 3’RACE PCR using primers designed from ZL.CYP.M and PL.CYP.M.One was 340bp from Lycopuslucidus turcz named ZL.CYP.3 and the other was 625bp from Eupatorium fortunei named PL.CYP.3.ZL.CYP.3 was one of Chlorophyll A-B binding protein in P450 super family whose function was not clearly. PL.CYP.3 was GASA superfamily protein in P450 super family which took part in the synthesine of GASA.更多还原