【作者】 朱斌；

【导师】 崔海瑞；

【作者基本信息】 浙江大学 ， 生物物理学， 2017， 硕士

【摘要】 农杆菌介导的遗传转化是广泛用于基因组分析的强大工具,也是目前获得转基因植物的主导技术。农杆菌介导的基因转移是极其复杂的生物学过程,需要许多农杆菌和植物的遗传因子协同参与下完成。T-DNA整合是农杆菌介导转化过程中最为关键的一步,越来越多的研究表明植物DNA损伤修复系统在对农杆菌T-DNA整合具有重要的作用,但主要关注的是DNA断裂修复基因,而对参与其它DNA损伤修复途径的基因尚未开展研究。为此,本文分析了不同DNA损伤修复途径基因对农杆菌浸染的响应,重点研究了水稻错配修复基因和碱基切除修复基因对T-DNA转移与整合的影响。主要结果如下:(1)DNA损伤修复相关基因对农杆菌浸染的响应。采用RT-PCR技术检测了NHEJ修复途径的基因ku70和lig4、HR修复的途径的基因rad51和recaa、MMR修复的途径的基因msh2和msh6、BER修复的基因fgp和maglp在农杆菌浸染后不同时间段的表达情况。ku70、lig4和rad msh2 和 msh6,ku70、lig4和 和maglp的表达水平分别在农杆菌浸染愈伤组织后12h、24h、36h和48h显著升高,这一结果说明这些DNA损伤相关基因可能参与了农杆菌介导的转化过程。(2)错配和碱基切除修复基因突变对转基因瞬间表达的影响。利用GUS组织化学染色法分析了与农杆菌共培养后愈伤组织中的gus基因瞬间表达频率。在错配修复基因(Os09g0407600)及碱基切除修复基因(Os04g0673400和Os09g0420300)的插入突变体与对照日本晴间,gus基因瞬间表达频率均无显著差异,说明错配和碱基切除修复基因突变不影响农杆菌介导转化的早期过程。(3)错配和碱基切除修复基因突变对T-DNA整合的影响。通过潮霉素抗性愈伤组织的筛选、分化和PCR分析了错配修复基因及碱基切除修复基因插入突变体愈伤组织及再生植株中的T-DNA整合。MMR基因突变体NF7784与NF9010的抗性愈伤筛选率和分化率及BER基因Os04g0673400突变体NC2747的分化率都呈现显著下降,但BER基因Os09g0420300突变体ND1052的抗性愈伤筛选率和分化率与对照无显著差异。对hpt和gus基因的PCR鉴定结果表明,MMR基因突变体的稳定转化率比对照下降49.33%～64.53%,BER基因插入突变体的稳定转化率则下降11.2%～36.53%。这一结果说明MMR基因和BER基因突变影响T-DNA的整合效率。更多还原

【Abstract】 Agrobacterium-mediated genetic transformation has been widely used as a powerful tool for genome analysis and the predominant technology for production of transgenic plants.The gene transfer mediated by Agrobacterium is a very complex biological process involving in the synergetic effect of genetic factors of both the bacterium and the host plant cell.T-DNA integration is the most critical step of the transformation process.More and more evidences suggest that plant DNA damage repair machinery plays an important role in Agrobacterium T-DNA integration.However,much more attention was paid to the genes related to DNA break repair and the genes in other DNA damage repair pathway awaits further investigation.The main results are as follows:(1)The response of DNA damage repair related genes to Agrobacterium infection.Expression of ku70 and lig4 in NHEJ repair pathway,rad51 and reca in HR repair pathway,msh2 and msh6 in MMR repair pathway,fgp and maglp in BER repair pathway was detected by RT-qPCR at different time after Agrobacterium infection.The expression level of ku70,lig4 and rad51,msh2 and msh6,ku70,lig4 and rec,fgp and maglp was significantly increased at 12h,24h,36h and 48h in callus,respectively.The results suggest that these DNA damage related genes may be involved in Agrobacterium mediated transformation process.(2)Effects of gene mutation in mismatch repair(MMR)and base excision repair(BER)pathway on transient expression of transgene gus.Transient expression frequency of gus gene in the callus after co-cultivation was analyzed by GUS histochemical staining method.There was no significant difference in the transient expression frequency between the MMR gene insertional mutant(NF9010 and NF7784)and the BER gene insertional mutant(NC2747 and ND1052)with the control Nipponbare,indicating that the early stage of Agrobacterium-mediated transformation is not affected by the mutations of MMR gene(Os09g0407600)and BER genes(Os04g0673400 and Os09g0420300).(3)The effect of MMR and BER gene mutation on T-DNA integration.The T-DNA integration was analyzed by screening and differentiation test of calli with hygromycin resistantce and PCR characterization of regenerated plants.The MMR gene mutant NF7784 and NF9010 showed significant decrease in screening rate of the resistant callus and differentiation rate of regenerated plants,the BER gene(Os04g0673400)mutant NC2747 also showed significant decrease in differentiation rate,but no differences in both screening rate and differentiation were detected between the BER gene Os09g0420300 mutant ND1052 and the control Nipponbare.Results of PCR characterization for hpt gene and gus gene showed that the stable transformation frequency for MMR mutants decreased by 49.33%～64.53%,while it decreased by 11.2%～36.53%for two BER gene mutants.These results suggest that the integration efficiency of T-DNA was affected by the mutations of MMR and BER genes tested in this research.更多还原