水稻叶片与颖花发育相关基因NRL2的克隆与功能分析

发布时间：2018-01-21 20:13

本文关键词： 水稻叶片发育厚壁组织细胞雄性育性基因克隆　出处：《中国农业大学》2016年博士论文　论文类型：学位论文

【摘要】：水稻是世界上重要的粮食作物之一,且水稻籽粒产量与叶片和颖花的发育密切相关。近年来,虽然许多叶片与颖花发育相关基因已成功分离并应用在水稻育种中,用以提高水稻产量。然而,其复杂的遗传调控机制仍不清楚。因此,进一步鉴定影响叶片与颖花发育的新基因,不仅能为水稻发育调控机制提供新的见解,还能为水稻株型与产量分子育种奠定基础。本研究采用图位克隆策略分离了水稻叶片、花药与籽粒发育相关的基因NRL2,并初步分析了其分子功能。主要结果如下：我们通过筛选水稻EMS突变体库,获得了2个叶片极度窄且半卷曲、雄性半不育、籽粒更加细长的等位突变体,命名为(narrow and rolled leaf 2-1) nrl2-1和nrl2-2。组织学分析发现,与野生型相比,nrl2突变体纵向叶脉数减少、远轴端厚壁组织细胞分化异常、绒毡层退化和小孢子发育异常。利用nrl2-1突变体与粳稻品种中花17杂交构建的F2分离群体,将NRL2基因精细定位于第3号染色体标记M4与M5之间,其物理距离约为59 kb。比较野生型与nrl2突变体精细定位区间DNA序列,我们发现LOC_Os03g19520在nrl2-1突变体的第9个外显子(+2662位置)存在一个单碱基G缺失,导致读码移框和编码蛋白的提前终止：而在nrl2-2突变体的第15个内含子3'剪接接收部位碱基G突变为碱基A,导致]mRNA剪切模式改变和蛋白编码提前终止。进一步的遗传转化证据表明LOC_Os03g19520即目的基因NRL2。酵母双杂交和双分子荧光互补实验表明,NRL2和RL14存在蛋白互作。测量剑叶次生细胞壁主要成分发现,与野生型相比,nrl2突变体叶片中的纤维素含量明显提高而木质素含量显著降低,与rl14突变体的变化一致。分析苯丙氨酸代谢产物发现,肉桂醇、杨梅素和二氢山奈酚的含量在nrl2突变体和nrl2/rl14双突中的含量显著降低。另外,苯丙氨酸代谢相关基因的表达水平在nrl2突变体与野生型之间也发生了改变,表明NRL2基因的突变影响了苯丙氨酸代谢途径,改变了木质素和黄酮醇的含量,进一步导致半卷曲的叶片和降低的雄性育性。另外,通过mRNA原位杂交和qRT-PCR分析我们发现,NRL2基因的突变影响了一些细胞分裂相关基因的表达。我们同时也发现,nrl2突变体颖壳的外厚壁组织细胞数比野生型要少,表明NRL2基因可能通过调控细胞增殖而调节叶片和籽粒的大小。而观察颖壳外厚壁组织细胞发现,突变体nrl2细胞数量较野生型显著减少,结果表明NRL2基因可能通过调控细胞增殖来调节叶片和籽粒大小。综上所述,NRL2基因是一个重要的发育调节因子控制基本的发育过程,是一个潜在的高产水稻育种目标。
[Abstract]:Rice is one of the most important food crops in the world, and the grain yield is closely related to the development of leaves and spikelets. Although many genes related to leaf and spikelet development have been successfully isolated and applied in rice breeding to increase rice yield, its complex genetic regulation mechanism is still unclear. Further identification of new genes affecting the development of leaves and spikelets can not only provide new insights into the regulatory mechanism of rice development. It can also lay the foundation for rice plant type and yield molecular breeding. In this study, the gene NRL2 related to rice leaf, anther and grain development was isolated by map cloning strategy. The main results are as follows: we obtained two allelic mutants with extremely narrow and semi-curly leaves, semi-sterile male and more slender grain by screening rice EMS mutants. They were named narrow and rolled leaf 2-1) nrl2-1 and nrl2-2.The results of histological analysis showed that they were compared with wild type. The number of longitudinal leaf veins of nrl2 mutant decreased, and the differentiation of thick-walled cells in the distal end of the mutant was abnormal. Nrl2-1 mutant and japonica rice variety Zhonghua 17 were used to construct F2 segregated population. The NRL2 gene was mapped finely between M4 and M5 on chromosome 3, and the physical distance was about 59 kb.Compared with the fine mapping region DNA sequence of wild type and nrl2 mutants. We found that LOC_Os03g19520 had a single base G deletion in the ninth exon (2662) of the nrl2-1 mutant. This led to the early termination of the reading frame and the coding protein: base G mutated to base A at the 3'splicing reception site of the 15th intron of the nrl2-2 mutant. The further evidence of genetic transformation showed that LOC_Os03g19520 was the target gene NRL2.yeast two-hybrid and bimolecular fluorescence. Complementary experiments show that. There was protein interaction between NRL2 and RL14. The main components of secondary cell wall of flag leaf were measured. It was found that compared with wild type, the content of cellulose in leaves of nrl2 mutant increased significantly, and the content of lignin decreased significantly. Analysis of phenylalanine metabolites showed that cinnamyl alcohol was found. The contents of myricetin and dihydrokaempferol in nrl2 mutant and nrl2/rl14 diploid were significantly decreased. The expression level of phenylalanine metabolism-related genes was also changed between nrl2 mutant and wild type, indicating that the mutation of NRL2 gene affected phenylalanine metabolism pathway. The content of lignin and flavonol was changed, which further led to semi-curly leaves and reduced male fertility. In addition, we found by mRNA in situ hybridization and qRT-PCR analysis. The mutation of NRL2 gene affected the expression of some genes related to cell division. We also found that the number of outer thick-walled cells in glume of nrl2 mutant was less than that of wild type. The results suggested that NRL2 gene may regulate the size of leaves and grains by regulating cell proliferation, while the number of nrl2 cells in the mutant was significantly lower than that in the wild type by observing the thick-walled cells outside the glume. The results showed that NRL2 gene may regulate leaf and grain size by regulating cell proliferation. In conclusion, NRL2 gene is an important developmental regulatory factor to control the basic development process. It is a potential breeding goal for high yield rice.
【学位授予单位】：中国农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S511

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