比较用质粒营救法和tail-pcr法获得水稻fsts的效率-comparing the efficiency of rice fsts obtained by plasmid rescue method and tail - pcr method.docxVIP

摘要获得突变体中T-DNA的旁邻序列是通过反向遗传学进行功能基因研究的重 要手段。实验比较了用质粒营救法和TAIL—PCR法获得T_DNA在水稻(Df归口 s口rfⅧ)基因组中旁邻序列的效率。实验构建了植株双元表达载体pBsBar，用根癌农杆菌09r06dc耙rf“Ⅲ，“晰咖cfP”妙EHAl05转入两个粳稻(ns口，fvP删，御D”fc“)品种州印on6口馏和中花1l的幼胚愈伤组织，共获得了1000株左右的转化苗。 用BASllA溶液筛选有85％左右的植株有抗性。利用质粒营救法获得T_DNA在 水稻中的旁邻序列效率可高达90％左右，而利用TAIL．PCR法的效率只有62％ 左右。质粒营救法和TAIL-PCR法获得的旁邻序列中是水稻基因组DNA的效率 分别是80％左右和89％左右。根据本实验结果，质粒营救法从转基因植株获得 的旁邻序列是水稻基因组DNA的效率是72％左右，比TAIL．PCR法要高l 8％左 右。根据获得的T-DNA在水稻基因组中的旁邻序列，T-DNA插入水稻基因组的 基因区和非基因区的几率相差不大，但是对于基因的5’．和3’．调控区有着一定 程度的偏好，而且T-DNA插入基因区时较多的是插在了内含子区。T．DNA插在 水稻染色体两臂的几率要远大于插在着丝粒，同时T_DNA也很少插在重复DNA 区域内，表明T_DNA难以插在水稻染色体的异染色质中。T-DNA在水稻基因组 插入位点附近的序列的AT含量主要在45％～65％左右，和水稻基因组AT含量 的趋势大致相同，说明了水稻基因组的碱基组成和T．DNA插入没有必然的联系。关键词：水稻(D，弦口删fvP)：■DNA；质粒营救法；1’AIL．PcRAbstractIt is imponant to emciently obtain narLking sequence Of T．DNA tags insertion in genome a strategy for inVestigating functional genomics by using reVerse genetics． In tllis study me two metllods were compared for their emciency to obtain thenallking sequences T-DNA tags insenion in rice((坳口J口“VP)genome．A pIantbinary vector pBsBar was constmcted atld traIlsf色rred into rice immature embryo callus by爿g，D6acfPrmm—mediated m砌10d．85 percem of about 1000 TO协msgenic plallts werc resistant to BASl=A solution．The result from 1 50 T0 T-DNA insenion1ines showed that也e emciency of plasmid rescue was about 90％，and that of 1’AIL·PCR was 62％．About 80％ofnanking sequence of T．DNAtags insertion in rice genome by plasmid rescue was rice genome，while about 89％by TAIL-PCR．As mentioned aboVe，me efficiency of obtaining nar山ing sequence of T_DNA tags insenion疳om transgenic plants by p1鹊mid rescue was about 72％，which was 1 8％ higher than t11at ofTAIL-PCR．We aIlalysis tlle obtaining nanking sequences of T-DNA tags insenion in rice genome by t、Ⅳo metllods．0ur results suggested tllat T-DNA insenions show a goodcOn℃lation w汕the predicted size diSt抽ution of genic and imergenic regions in therh genome and were evenly spread within genes，while me preference insenions in the 5’一a11d 3’一rcgul