高通量转化高粱cDNA文库改良水稻

发布时间：2018-07-04 22:31

本文选题：水稻改良 + 突变体库　；参考：《武汉大学》2016年博士论文

【摘要】：水稻产量关乎到世界粮食安全,而且提高水稻产量是科学研究的热点。随着水稻基因组DNA的测序完成,更多的水稻农艺性状相关的功能基因被鉴定了,而这些被克隆的水稻农艺性状相关的基因多被应用于传统的水稻育种工作中。高粱、玉米和小麦都是禾本科中的谷类作物,携带着许多非常有价值的基因,可以用其改良水稻。为了探索高粱中有用的基因,我们构建了一个高粱cDNA过表达文库,并对水稻进行高通量的转化,以期望获得一些产量高的、抗病强的、能适应极端环境的等有价值的水稻转基因系。本研究利用构建好的高质量的高粱cDNA过表达文库转化水稻,创建了上千株水稻突变体。在高粱cDNA过表达文库被应用于转化以前,我们对其进行了质量评价,37个cDNA中有20个cDNA具有和NCBI数据库里一致的CDS,另外16个也具有ORF,这说明文库的质量符合要求；同时,通过同源比对,我们也发现了5个高粱新基因。转基因获得1229株TO代水稻转基因系,性状涉及株高、分蘖、叶型、叶色、叶夹角、穗型和粒型等,我们对这些性状进行世代的观察和相关数据收集,结果是92.2%的叶长发生了变化,58.3%的叶宽有差异,48.6%的抽穗期有变化,近91.5%的株高变矮和83.9%的有效穗有明显不同：通过对所有转基因系的性状考察,转基因过程确实影响了水稻的株高和育性；针对稳定遗传的转基因系比如叶夹角、穗子大小、粒型(和淀粉合成)等,我们可以设计实验方案进行下一步的研究。对0752株系的基因Sb01g013740进行克隆并重新转化水稻,我们证明了此基因过表达能够使水稻种子变小,而对于该基因是否与水稻株高和穗型相关,这需要进一步的分析。由于产量性状也是我们关注的焦点,所以我们就对92个株系进行了考种分析,结果是获得了19个产量增加的株系,并克隆了14个基因,而这些基因有13个具有和NCBI数据库里一致的CDS,其中7个CDS和高粱抗逆性有关,这暗示我们这些基因很可能通过增强水稻的适应性来提高水稻产量。本研究是对利用cDNA过表达文库创建水稻功能获得性突变体的一种尝试,事实证明,此方法是可行的、有效的。
[Abstract]:Rice yield is related to world food security, and increasing rice yield is a hot spot in scientific research. With the completion of rice genomic DNA sequencing, more functional genes related to agronomic traits have been identified, and the cloned genes related to agronomic traits have been widely used in traditional rice breeding. Sorghum, corn and wheat are cereal crops in the family Gramineae, carrying many valuable genes that can be used to improve rice. In order to explore the useful genes in sorghum, we constructed a sorghum cDNA overexpression library, and carried out high-throughput transformation of rice, in order to obtain some high-yield, disease-resistant, An equally valuable transgenic rice line that adapts to extreme conditions. In this study, a high quality sorghum cDNA overexpression library was constructed to transform rice into rice, and thousands of rice mutants were created. The quality of sorghum cDNA overexpression library was evaluated before it was used for transformation. Twenty of the 37 cDNA had CDSs consistent with the NCBI database, and the other 16 had ORFs, which indicated that the quality of the library met the requirements. We also found five new sorghum genes by homologous comparison. 1229 transgenic lines of to generation were obtained. The characters were related to plant height, tiller, leaf type, leaf color, leaf angle, panicle type and grain type. The results showed that 92.2% of leaf length had been changed. 58.3% of leaf width was different, 48.6% of heading date was different, 91.5% of plant height was shorter and 83.9% of effective panicle was different. The transgenic process does affect the plant height and fertility of rice, and we can design experiments to further study the stable genetic transgenic lines such as leaf angle, ear size, grain shape (and starch synthesis). The gene Sb01g013740 of 0752 strain was cloned and retransformed into rice. We proved that overexpression of this gene can make rice seeds smaller, but whether the gene is related to plant height and panicle type of rice needs further analysis. Because yield traits are also the focus of our attention, we analyzed 92 lines, and as a result, we got 19 lines with increased yields and cloned 14 genes. Thirteen of these genes have CDSs consistent with the NCBI database, 7 of which are related to sorghum stress resistance, suggesting that our genes are likely to increase rice yield by enhancing rice adaptability. This study is an attempt to create rice functional acquired mutants by using cDNA overexpression library. It has been proved that this method is feasible and effective.
【学位授予单位】：武汉大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S511

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