【作者】 刘娜；

【导师】 张宪省； 张翔宇；

【作者基本信息】 山东农业大学 ， 发育生物学， 2020， 硕士

【摘要】 植物激素参与调控植物的生长发育。植物甾醇类激素,包括植物甾醇和油菜素内酯(Brassinosteoids,BRs)。油菜素内酯作为一种重要的植物激素,参与了许多生物过程的调控。油菜素内酯合成途径中的限速酶C-22羟化酶DWF4(CYP90B1),通过调控植物内源油菜素内酯的水平,影响植物农艺性状的发育。玉米(Zea mays L.)是重要的粮食、饲料作物,也是我国第一大作物。本实验室前期利用农杆菌介导的基因转化技术,获得了3个稳定的过表达ZmDWF4的转基因株系L100、L166、L225。本研究在此基础上,利用这3个农艺性状优良的转基因株系配制了27个杂交组合,分析了ZmDWF4基因过表达对玉米杂交种产量的影响。研究结果和结论如下:(1)通过Western Blotting和激素测定,进一步证实了过表达转基因株系中目的基因得到了高水平表达。(2)过表达ZmDWF4能够促进种子发育,转基因株系的籽粒变大,穗粒数增加,粒重增加,单穗产量提高。果穗的穗粒数增加显著,平均上升了15%。单穗重量增加显著,转基因株系L100、L166及L225相比野生型产量分别增加了41.6%、20%及11%。(3)过表达ZmDWF4能够促进氮响应基因及氮转运基因的高水平表达,从而促进了玉米对硝态氮的吸收。(4)过表达ZmDWF4能够促进转基因植株生长。在苗期,转基因株系生长旺盛;在植株成熟期,转基因株系相比野生型农艺性状得到改良,叶片数目增多,穗位叶叶面积增大,光合能力增强,衰老延迟。(5)转基因株系L100的叶片表达谱分析发现,有161个基因发生上调表达,140个基因下调表达,差异表达基因的功能聚类主要集中在植物发育的细胞壁,细胞分裂及增殖的途径和光合相关途径。在转基因株系L100的授粉后10天的种子的转录组数据中,272个基因上调表达,191个基因下调表达,在授粉15天的转录组数据中,437个基因上调表达,238个基因下调表达。对两组表达谱的差异基因的功能聚类分析发现差异表达基因主要富集到代谢相关的途径,其中糖代谢及蛋白质代谢途径相关基因大部分发生上调表达。(6)过表达ZmDWF4能够增加杂交种产量。小区测产发现转基因的杂交种产量相比野生型杂交种平均增加了17.25%。综上所述,过表达ZmDWF4能够提升BRs水平,调控生长及代谢相关途径的基因表达,改良转基因株系的农艺性状,增加了杂交种的产量,为玉米的遗传改良提供了一种有效的生物技术策略。更多还原

【Abstract】 Plant hormones regulate plant growth and development.Plant sterol hormones include phytosterol and BRs(Brassinosteoids).Brassinosteoids,an important plant hormone,regulate many processes in plant growth and development.DWF4(CYP90B1),encoding C-22 hydroxylase,can affect the development of agronomic traits of plants by regulating the levels of endogenous BR.Maize(Zea mays L.)is an important grain and feed crop,and a crop with the largest area in China.In the early study,using Agrobacterium-mediated genetic transformation technology,we obtained three stable overexpressed ZmDWF4 transgenic plants L100,L166,L225.Here,using the three transgenic lines with excellent agronomic traits to do 27 high productive crosscombinations,we analyzed the effect of ZmDWF4 overexpression in maize hybrids.The results and conclusions are as follows:(1)By Western Blotting and hormone assay,it was further confirmed that the target gene was highly expressed in the overexpressed transgenic lines.(2)Overexpression of ZmDWF4 can promote seed development.The kernels size,kernel number per ear,grain weight and ear weight were all increased in transgenic lines compared to the wild type Qi 319.The kernels number of ear significantly increased by 15% on average.The ear weight in transgenic lines L100,L166 and L225 was increased by 42.7%,19.2% and 12%,respectively,compared to that in the wild type.(3)Overexpression of ZmDWF4 can promote the high expression levels of the response genes and transport genes of nitrate nitrogen and can promote the absorption of nitrate nitrogen in transgenic lines.(4)Overexpression of ZmDWF4 can promote the transgenic lines development.At the seedling stage,the transgenic lines developed more vigorous than the wild type.At the adult stage,the agronomic traits were improved in transgenic lines.The transgenic lines showed increased leaf number,the ear leaf area,and thephotosynthetic capacity.(5)The leaf wide-genome expression profiles data showed 301 different expression genes including 161 up-regulated expression and 140 down-regulated expression in the transgenic line L100 and wild type Qi 319 inbred.The significant GO enrichment mainly showed cell,development and growth related terms and light related terms.Through transcript profile analysis in seeds at 10 days after pollination,we identified a total of 463 differentially expressed genes,and of them,272 were upregulated,and 191 were downregulated.Transcriptome data in seeds of 15 days after pollination showed 675 differentially expressed genes,and of them,437 were upregulated,and 238 were downregulated.The functional cluster analysis of different expression genes demonstrated that they were mainly involved in cell metabolism in two RNA-seq data,especially the expression of starch and protein synthesis genes that were increased compared to the wild type.(6)Overexpression of ZmDWF4 can improve the hybrid yield traits,and increase the hybrid yield.By analysis of plot experiments,transgenic hybrids were increased by 17.25% on average compared to wild type hybrids.Taken together,the expression of ZmDWF4 can increase the BRs level to regulate development and metabolism-related gene expression,which can improve the agronomic traits of transgenic plants,and increase the yield of hybrid.The study provides a useful biotechnology strategy for the genetic improvement and further breeding of maize.更多还原