【作者】 龙国徽；

【导师】 申斯乐；

【作者基本信息】 吉林大学 ， 生物化学和分子生物学， 2005， 硕士

【摘要】 高压技术就是近年来发展起来的一种全新的方法与手段,物理方法简单、易行、普遍适用,在实验环境下很容易实现。同时,压力在研究生物方面获得的信息量大,研究范围广而在国际上得以迅速发展。大麦是世界上重要的粮食作物之一,在大麦遗传育种中应用DNA 分子标记RAPD(random amplified polymorphic DNA)和ISSR(inter simple sequence repeat)技术,对高压诱导的变异材料和原种之间的进行多态性进行分析,从DNA 水平上寻找它们间的差异,对部分RAPD 和ISSR 产生的特异片段克隆测序和DNA 序列分析,进一步探讨高压诱变育种中所涉及的DNA 变化的分子机理。利用不同静水压力和不同保压时间处理萌动的大麦种子,随着压力的增加和保压时间的延长,发现高压对大麦种子萌发及幼苗生长有一定的抑制作用,并且压力及保压时间对种子萌发均有影响,大麦发芽率和成苗率明显下降。静水高压能够显著降低大麦的种子的发芽率和成苗率,高压处理后的大麦在苗期的生长受到明显抑制。用RAPD和ISSR 技术对不同静水压力和不同保压时间处理的大麦的遗传多样性进行了分析,21 个ISSR 引物和19 个RAPD 引物分别扩增出765 和454条带,选用甲基化敏感的内切酶HpaII 和MspI 分别对的基因组DNA 进行酶切,不同引物之间及不同甲基化酶酶切都观察到一致的甲基化变异。利用ISSR 和RAPD 技术从高压处理后的大麦的DNA 中得到8 个特异扩增片段,回收该特异扩增片段并将其克隆到pGEM-T Easy 载体上进行序列测定,进行BLAST 分析。序列信息结果表明8 条测序带有2 条找到同源序列,而且两条特异条带的同源性序列是同一克隆序列(Hordeum Vulgare BAC 克隆未知基因)不同区段,序列高度同源。更多还原

【Abstract】 Barley seeds were treated by different high hydrostatic pressure or were kept for different time. As comparison to control, both germination rate and seedling rate decreased considerably with pressure or treated time increasing . The above results showed that high hydrostatic pressure could affect the seeds germination ,seedling growth.. Plant growth was inhibited in treated materials during the early growth stage of seedling. The genetic variation of the different high hydrostatic pressure and the different time treated material were investigated by ISSR and RAPD analysis, 20 ISSR and 19 RAPD primers generated 937 and 600 bands, of which 382(40.76%) and 266 (44.33%) were polymorphic respectively .For T2 and P3,173 and 187 bands were generated with 66.21% and 68.77% being polymorphic. ISSR analysis indicated that the mean absolute distance coefficient between the original breed and the T2 is 0.6047, while that between the original breed and the P3 is 0.5636. RAPD analysis demonstrated that the mean absolute distance coefficient between the original breed and the T2 is 0.6168, while that between the original breed and the P3 is 0.5949. Some specific amplified DNA fragments was obtained from genomic DNA of high-pressure induced mutagenized barley using ISSR and RAPD technique.After cloning this fragment into p GEM-T easy vector and sequencing it, we found that ISSR and RAPD analysis of these two mutant and the original breeds revealed that there are significantly polymorphism between the two mutants and the original breed, each with 58.59% and 49.93%. DNA methylation is the main cause of transcription algensilencing. ISSR analysis indicated that there existed significant difference in DNA methylation status between the original breed and the high hydrostatic pressure treated. Comparison of the eight clonging sequences with Hordeum vulgare sp. vulgare cultivar Morex BAC clone 773k14,更多还原