【作者】 付金锋；

【导师】 季良越；

【作者基本信息】 河南农业大学 ， 作物遗传育种， 2004， 硕士

【摘要】 对转Bt基因材料杂交种NC7882的F2,F3,F4后代和自交系C237采用卡那霉素叶片涂抹、潮霉素叶片涂抹、田间接虫鉴定和NOS终止子序列引物和Bt序列设计的引物（W）PCR分子鉴定等5种方法的鉴定，并对鉴定结果进行成对数据T测验。两对引物的PCR分子鉴定结果表明叶片总DNA扩增产物的条带在预期扩增范围内，说明转基因后代中外源Bt基因以及NOS终止子的存在。以W为引物的分子鉴定结果与田间接虫鉴定结果T测验之间不存在显著差异，说明以W为引物的分子鉴定结果的可靠性，可以作为标准鉴定方法。以NOS为引物的PCR鉴定结果与以W为引物的PCR鉴定结果进行成对数据T测验结果表明两者之间存在显著差异，说明以NOS为引物的分子鉴定结果的不可靠，这可能是因为用NOS引物扩增产物片断小，琼脂糖凝胶电泳不能区分分子量稍有差异的扩增片断与DNA碎片。1000mg/L（处理1）和（处理2）1250mg/L浓度的卡那霉素鉴定结果与以W为引物的分子鉴定结果成对数据T测验也存在显著差异，而500mg/L（处理3）浓度的潮霉素鉴定结果以W为引物的分子鉴定结果成对数据T测验不存在显著差异，说明转Bt基因玉米NC7882和C237含有抗潮霉素标记的基因，并能表达,而不含抗卡那霉素基因，或者抗卡那霉素基因不能表达，因而潮霉素叶片涂抹的方法可以作为是否携带Bt基因间接鉴定方法。对杂交种NC7882杂交2代用W引物PCR鉴定结果阳性阴性植株的分离比例符合3：1，F3、F4代株系大部分也符合一对显性基因的分离比例，验证转Bt基因的遗传是符合一对显性基因的孟德尔分离规律，Bt基因可以在世代中稳定遗传。通过对转基因玉米后代不同株系抗虫性的筛选，为以后抗虫性植株的选育打下良好的基础。更多还原

【Abstract】 The insect-resistance trait of F2,F3,F4 progeny from transgenic Bt hybrids NC7882 and transgenic Bt inbred lines C237 were identificated by five differ- -ent methods as kanamycin identification, hygtomycin iden tific at ion, identification of insect -resistance in field and PCR analysis. PCR primers were designed according to sequence of Bt gene and NOS terminator,The amplify- -ing results by two pairs of primers PCR revealed that the length of amplified flagment was consistant with the length expected. This indicated that the Bt gene and NOS terminator were existed in the progeny of NC7882 and C237. T-test of pairs data showed there was no obvious difference between the result of insect -resistance identification in field and that of W primer PCR identifica- -tion,which was regarded as standard identification technique . There was obv- -ious difference between the result of NOS primer PCR identification and that of W primer PCR identification,So the result of NOS primer PCR identificati- -on was truthless.Because NOS primer PCR anplified flagment was not enough long,it was not easy to distinguish the amplified flagment from small DNA piece by gelose electrophoresis . There was significant difference between the results of kanamycin identification and that of W primer PCR identification, but there was no significant difference between identifying result of hygtomycin with 500mg/L concentration and that of W primer PCR.This indicated that the anti-- hygtomycin gene was existed and express, anti-kana- -mycin gene was not existed or not express in transgenic Bt hybrids NC7882 <WP=44>and transgenic Bt inbred lines C237. Thus the method about hygtomycin涂抹(daub) leaf can indirectly identify Bt gene . In NC7882 and C237 progeny the segregation of Bt+and Bt-plants fitted 3:1 in NC7882F2and parts of F3, F4 linesally ,which showed one pair dominant gene Mendel inheritance.Those researchs about identification methods and selection insect-resistance lines in Bt transgenic Progeny make a solid base for insect-resistance.breeding.更多还原