【作者】 杨永智；

【导师】 路铁刚；

【作者基本信息】 中国农业科学院 ， 生物化学与分子生物学， 2004， 硕士

【摘要】 水稻(Oryza sativa L.)是最重要的粮食作物之一，具有较小的基因组(430Mb)，与其他禾本科植物的基因组具有良好的共线性，易于体外操作与分化，是研究单子叶植物基因组的模式植物。近年来水稻基因组研究取得了很大进展，构建了遗传图谱和物理图谱，完成了籼稻和粳稻的全基因组草图测序(Yu et al.,2002;Goff et al.,2002)，2002年12月18日国际水稻基因组测序计划工作组在东京宣布，国际水稻基因组测序计划已圆满完成，共测定碱基对366，000kb，精确度达到99.99％，并预测遗传基因62，435个。在此基础上，许多实验室开始大规模地、系统地进行水稻基因功能的研究。建立水稻突变体库是功能基因组学研究的有效手段，而高效的水稻转化体系是建立突变体库的重要基础。本研究正是在建立起了高效水稻农杆菌转化体系基础上，构建了一个大规模的水稻增强子捕获群体，并对其进行了分子鉴定，初步建立起适合该群体的扩增T-DNA侧翼序列的PCR-walking技术体系，已扩增了部分突变体基因组中的T-DNA侧翼序列，并对部分侧翼序列进行了测序分析。 本研究是国家“863”计划项目“水稻突变体库的建立”的一部分，在这两年的研究中，在与实验室全体人员的共同努力下，取得了以下几个方面的进展： 1．在农杆菌(Agrobacterium)介导的水稻转化中，发现共培养后的愈伤组织在选择培养基上的继代培养的时机对抗性愈伤组织产生和正常生长有很大的影响，最佳的时机是在选择培养10～15天后进行继代培养，这样90％以上的愈伤组织可以产生能健康生长的抗性愈伤组织；发现抗性愈伤组织在选择培养基上的继代培养时间对后边的分化效率有至关重要的影响，抗性愈伤组织继代培养4～12天可以有比较高的分化率，超过16天或少于2天，分化率急剧降低。抗性愈伤组织在选择培养基上培养8天时，分化率最高，达到85％；在胚性愈伤的诱导、共培养侵染菌液的最佳浓度的确定、共培养的时间以及转化不同阶段的最适培养基类型等方面的研究分析基础上，优化建立起一套高效、操作性强、适合水稻粳稻品种日本晴(O.sativa Spp.Japonico)的农杆菌转化方法体系。从愈伤组织诱导到得到水稻转化体只需2.5月左右的时间。 2．选用增强子捕获质粒(Enhancer-trapping vector)pFX-E24.2-15R，以粳稻日本晴为实验材料，利用农杆菌介导的方法，获得了65，707个增强子捕获T-DNA插入标签系。经PCR和Southern杂交检测，表明95％以上的转化体含有T-DNA插入片段。对T0代植株进行了Southern杂交分析，T-DNA插入片段的单拷贝的植株约占43％，平均拷贝数为1.7。 3．对11，560株T0代水稻转化植株抽穗期的叶和未成熟种子进行了GUS组织化学染色，GUS基因在抽穗期种子部位和叶部的总表达率为27.23％，在叶部的表达率为10.07％，在胚的不同部位表达率为11.01％，在种皮的表达率为8.28％，在胚乳的表达率为3.42％。筛选出210个在这一生育期表达活性很强的增强子捕获系，取样并提取了DNA，以做进一步的研究。 4．用PCR Walking扩增了328个筛选出的突变体基因组的T-DNA侧翼序列，对其中82个序列进行了测序，作了初步分析。初步建立起突变体插入序列侧翼序列信息的获得、分析等方法体系，已获得一些有价值的T-DNA插入位点信息。更多还原

【Abstract】 Rice (Oryza saliva) is one of the most important food crops. It is also a good model for studies of monocot plants because of its relative small genome (430Mb), syntenic relationship with other agronomically important cereal species, the availability of genome resources such as well-defined genetic and physical maps, well-rounded transformation system and the gene draft sequence of Japonica cv. and Indica cv. Moreover, IRGSP has declared on December 18, 2002 at Tokyo that the plan of IRGSP has been finished with 366,000 kb DNA sequenced totally, the accuracy reached at 99.99% and 63,435 genes have been predicted. Large-scale and systematic functional genome studies of rice have been performed in several laboratories based on the above achievements. Construction of rice mutant bank is an efficient tool for functional genome research while efficient transformation system composed the basis for it. In this study, a high efficient Agrobacterium-mediated transformation system has been established and a large-scale rice enhancer trapping population has been generated. Further, molecular definition for this population has been done and PCR-walking adapted to this population for the amplification of T-DNA flanking sequence has been set up, simultaneously, we have analyzed some flanking sequence.This study is one part of "rice mutant bank construction" sponsored by The National High-Tech Program (863 program). Several progresses have been achieved with the efforts of all the members of our team within 2 years. Detailed information presented as follows:1. tn our studies, we have found that the timing of subculture on selection medium after coculture has prominent effects on the generation of resistant calli and their normal growth. Subculture after 10-15 days selection has been confirmed as the best time and over 90% calli could generate resistant calli under this status which were growing fine. We also found that the subculture interval under selection has momentous effects on the frequency of later differentiation. They have higher frequency of differentiation if 4-12 days subculture on selection medium were chosen while their frequency of differentiation decreased sharply if the subculture interval is over 16 days or less than 2 days. As an example, the frequency of differentiation reached at 85% if resistant calli were subcultured for 8 days on selection medium. We have set up and optimized one set of transformation system with characteristics of high efficiency, easily being performed and adapted O. sativa spp. Japonica. This integrated the achievements on the induction of embryogenic calli, the determination of the most optimal concentration of inoculation Agrobacterium solution, the interval of coculture with Agrobacterium and selection of different medium types at different stages. It takes only 2.5 months from induction of calli to generation of transformants.2 65,707 T-DNA tagged enhancer-trapping lines have been obtained through Agrobacterium- mediated transformation by applying enhancer-trapping vector pFx-z24.2-15R and employing rice (O. Sativa Spp Japonic) as material. It is indicated that more than 95% transformants harbored T-DNA insertion fragment confirmed by PCR and Southern blot analysis. In all TO transformants, there are about 43% events has one copy of T-DNA insertion and the average copy number of T-DNA insertion is about 1.2 confirmed by Southern Blotting. 3, The histochemical staining of GUS activity has been performed on the leaves and immature seeds of11,560 TO lines at heading stage. The general GUS expression frequency in seeds and leaves are 27.07% while 11.01% in the different parts of embryo, 8.28% in seed capsule and 3.42% in endosperm. 210 enhancer trapping lines with strong expression at this developmental stage have been selected form this population. The samples have been harvested and the DNA has been extracted for further studies.4. The T-DNA flanking sequences of 328 mutant lines have been amplified by PCR-walking. 82 lines of this group have been sequenced and analyzed pr更多还原