抗纹枯病基因的克隆及转基因小麦的研究-作物遗传育种专业毕业论文.docxVIP

抗纹枯病基因和特异性启动子的克隆及其转基因小麦的研究 specific primers of expression vector gene to conduct PCR detection for the living transformed plants, we obtained 12 plants that PvPGIP2 and TaLTP4 presented positive, and the conversion rate was 0.6%. The RSS1P::TiERF1 transgenic plants in T0 and T1 generations were subjected to PCR, PCR-Southern, RT-PCR, and Q-RT-PCR analyses, and Rhizoctonia cerealis resistance tests. The molecular detection results showed that the transgene RSS1P::TiERF1 was introduced into Yangmai 12 and was inheritable. In the RSS1P::TiERF1 transgenic wheat plants, TiERF1 expressed in root, stem, and leaf except seed. The highest expression level was observed in root. Compared to the untransformated Yangmai 12, resistance to R. cerealis in the RSS1P::TiERF1 transgenic wheat plants was obviously improved, which was similar to that in the Ubi::TiERF1 transgenic wheat plants. The agronomic traits of RSS1P::TiERF1 transgenic plants had no obviously changed compared to untransformated Yangmai 12. These results suggest that RSS1P promoter is feasible to be used for developing new transgenic wheat germplasm. Through analysis and verification on several constructed promoters by GUS staining technique, it showed that Intron had an enhance effect on promoter RSS1P, and expression quantity of RSS1P2897 and RSS1P1715 in callus was extremely low as well as sole Intron possessed the ability of promoter. PCR detection was carried on DNA of callus , and it showed that bombardment expression vector had been into callus. Key words: Phaseouls vulgaris(PvPGIP2); Triticum aestivum(TaLTP4); RSS1P; Ubiqiutin; gene clone; tissue-specific expression; transgenic wheat; transient expression 4 符号说明 英文缩写 Amp 英文名称 ampicillin 中文名称 氨苄青霉素 bp base pair 碱基对 BSA bovine serum albumin 牛血清蛋白 cDNA complementary DNA 互补 DNA CTAB Cetyl Trimethyl Ammonium Bromide 十六烷基三甲基溴化铵 ddH 2O distilled and deionized water 重蒸水 DEPC diethyl pyrocarbonate 焦碳酸二乙酯 DNA deoxyribose nucleic acid 脱氧核糖核酸 dNTP deoxynucleosi