【作者】 刘志华；

【导师】 詹亚光；

【作者基本信息】 东北林业大学 ， 林木遗传育种， 2002， 硕士

【摘要】 应用农杆菌介导法向白桦(Betula platyphylla Suk.)基因组中导入抗虫基因(蜘蛛杀虫肽与Bt基因C肽序列连接在一起表达的一种融合蛋白基因)，并首次获得抗虫转基因植株。为白桦的遗传改良与高新技术育种开辟了一条新途径，也为其他林木树种的遗传改良提供了理论依据。 利用双元载体的根癌农杆菌LBA4404菌株(Agrobacterium tumefaciens)，含质粒pYHY(目的基因及nptⅡ、gus基因)，对白桦试管苗茎段，叶柄，叶片三种外植体进行侵染，结果表明：大叶片生长势强，为转基因的最优外植体，转化率能够达到22％。转基因卡那霉素抗性植株中，GUS表达阳性率为43％，随机抽样的方法进行杀虫肽和nptⅡ基因的PCR检测阳性率均为100％。 经过分子生物学检测表明，Southern斑点杂交76.5％植株表现阳性。转基因植目的基因(蜘蛛杀虫肽+Bt基因的C肽)的PCR Southern杂交阳性率为87.5％。经过Southern印迹杂交47.2％的GUS阳性苗杂交呈阳性，说明外源基因已稳定整合到白桦基因组中，多次继代后，仍很稳定。Northern斑点杂交表明，外源基因在转基因植株中正常表达。 转基因植株的杀虫实验结果表明，转基因植株的杀虫率能达到30％以上。舞毒蛾幼虫发育推迟，15天时处理为4龄，但对照仍为2龄，发育推迟了2个龄期；舞毒蛾生长受到抑制，对照的体重为处理的9.7倍与6.4倍，外源基因严重抑制幼虫的生长，说明外源基因在转基因白桦中能够正常表达，转基因植株表现一定的抗虫性。更多还原

【Abstract】 The paper first reported that transgenic plants of Betulla Platyphylla were obtained by Agrobacterium mediated method in China. It replenished the blankness of Birch genetic transformation, and provided a new path for genetic improvement in this species.The materials as explant in transformation come from Birch leaf, stem segment and leaf stalk, and the Spider toxin gene was used as foreign gene for this transformation experiment. It showed that the best explant was the big leaf, on which the transformation frequency was 22%. By GUS detection, there were 43 percent of the plants with kanamycin resistance, and 100 percent of positive result, by PCR amplification, was gotten from random sampling.It indicated that 76.5 percent of positive frequency of all the plants was achieved by using Southern dot hybridization, 87.5 percent of the plants with GUS positive activity was gotten by PCR southern blotting and 42 percent of the plants with GUS positive activity was gotten by southern blotting. It means that insect resistant gene was transferred and integrated into genome of the plants. By northern dot blotting, it also showed that the foreign gene could express in the transgenic plants.The insect feeding test indicated that the development of Lymantria dispar was clearly put off, in which the insects feeded transgenic plants were 2 ages, when the insects as the control were 4 ages after treating 15 days. Besides, the insect growth was restrained, that the weight of insect as control was 9.7 and 6.4 times compared to the treated ones. It also resulted that the foreign gene could normally express in the transgenic plants by displaying insect resistance in a certain degrees. It reached to 30 percent in mortality rate to Lymantria dispar.更多还原