水稻控制花粉管生长基因OsCNGC13的图位克隆及功能分析和水稻卷叶基因OsZHD1的功能研究

发布时间：2018-08-11 08:31

【摘要】：小穗育性是水稻高产的决定因素之一。高且稳定的小穗结实率是水稻超高产杂交水稻的重要特征。水稻半不育突变体在作物育种、遗传学、生殖生物学和分子生物学的研究中提供了很好的材料,若能用稳定的半不育突变体进行研究,阐明半不育产生的分子机制,对克服生产中遇到的半不育问题具有重要的借鉴意义。在开花植物受精过程中,花粉管在雌蕊中生长的过程就是其与雌蕊相互识别的过程,为了更好地理解花粉管和雌蕊的相互作用分子机制,需要识别和分离涉及此过程各个阶段的基因,并对其功能进行分析。作为进行光合作用的主要器官,叶片对于种子植物的生长发育十分重要。在水稻育种中,倒三叶叶片长、窄、薄、直立且适当卷曲是超高产杂交水稻的重要特征。其中,叶片适当卷曲是突破产量瓶颈的关键,因此,克隆控制卷叶基因并明确其功能具有重大的生产意义。本研究对一个新的小穗半不育突变体进行研究发现,在水稻受精过程中,OsCNGC13能够促进花粉管通道内的细胞程序性死亡而促进花粉管的生长,该结果为进一步阐明水稻生殖发育的分子机制提供了新思路。此外,对一个新的水稻卷叶突变体进行研究发现,OsZHD1在水稻形态建成尤其是泡状细胞的形成和分布中起重要作用,过表达该基因会导致植株叶片卷曲,泡状细胞数量增多且排列异常。本研究主要结果如下:1.ORF1突变是导致突变体结实率下降的主要原因。细胞学观察表明,突变体花器官形态、花粉粒发育和体外萌发均正常;而在花粉管生长和胚囊极核位置方面表现异常。遗传分析表明,该突变由单显性基因控制。通过精细定位将该基因定位在52-kb范围内。基因组测序结果表明突变体中有44-kb片段发生了倒置,两边的断口分别在ORF1和ORF3的内部,使得ORF1被截短形成NewORF1。3'RACE实验表明NewORF1与野生型ORF1相比,序列差异表现在C端。通过转基因互补发现,野生型中表达NewORF1会使植株结实率下降;在W109sss1-D中过表达ORF1,转基因植株结实率有所提高;利用Crispr技术将野生型中ORF1进行基因敲除,全部八个阳性家系植株的结实率都有不同程度的下降。2. ORF1 编码一个环核苷酸门控离子通道(cyclic nucleotide-gated channels,CNGCs)蛋白OsCNGC13,该蛋白主要通过调节花柱内细胞程序性死亡而控制小穗育性。时空表达分析发现OsCNGC13呈组成型表达模式,并在雌蕊中大量累积。水稻和烟草系统表明该蛋白亚细胞定位于细胞膜。电生理实验证明OsCNGC13是一个钙离子通道蛋白。水稻中有3个钙调蛋白且均能与OsCNGC13互作,但在水稻16个CNGC家族成员中并未发现能与OsCNGC13互作的同源蛋白。利用原子吸收能谱对开花前后雌蕊中钙离子相对含量进行分析,发现在9311花柱中钙离子明显积累,而在突变体中并无此种现象。此外,用阿新蓝染料对成熟花柱进行染色,发现突变体较野生型着色较浅且范围较小。在开花后30分钟时,能观察到9311花粉管通道中的死亡细胞,但是突变体中相应部位的细胞依旧完整。开花后两小时,能观察到9311花粉管通道中存在大量细胞间隙,但是在突变体中只有少量细胞发生了消亡。3. OsZHHD1控制水稻叶片卷曲。对反卷叶突变体进行细胞学观察发现,突变体叶片中泡状细胞增加且排列异常。遗传数据和分子实验表明,T-DNA插入使锌指同源异型盒转录因子OsZHD1过表达,从而导致水稻叶片出现反卷。OsZHD1表现为组成型表达并在发育中的叶和穗子中积累。野生型中过表达OsZHHD1发现在所得到的转基因阳性家系中,均出现了反卷叶的表型。
[Abstract]:Spikelet fertility is one of the determinants of high yield in rice. High and stable spikelet setting rate is an important characteristic of super high yield hybrid rice. The molecular mechanism of Semisterility in flowering plants is of great significance for overcoming the problem of Semisterility in production. In the process of fertilization, the growth of pollen tube in pistil is the process of recognition between pollen tube and pistil. As the main organ of photosynthesis, leaf is very important for the growth and development of seed plants. In rice breeding, long, narrow, thin, erect and properly curled leaves are the important characteristics of Super-high-yielding hybrid rice. In this study, a new spikelet semi-sterile mutant was studied. OsCNGC13 could promote programmed cell death in the pollen tube pathway and promote the growth of pollen tube during rice fertilization. The results are as follows:1. In addition, studies on a new rice leaf roll mutant have shown that OsZHD1 plays an important role in rice morphogenesis, especially in the formation and distribution of vesicular cells. Overexpression of OsZHD1 results in leaf curl, increased number and abnormal alignment of vesicular cells. The main results were as follows: 1. ORF1 mutation was the main reason for the decrease of seed setting rate. Cytological observation showed that the mutant had normal floral organ morphology, pollen grain development and germination in vitro, but abnormal pollen tube growth and polar nucleus position of embryo sac. Genetic analysis showed that the mutation was controlled by a single dominant gene. The result of genome sequencing showed that 44-kb fragment was inverted in the mutant, and the fragments on both sides were located in ORF1 and ORF 3 respectively. The result of the ORF1 truncated to form NewORF1.3'RACE showed that the sequence difference between NewORF1 and ORF1 was C-terminal. Over-expression of ORF1 in W109sss1-D increased the seed setting rate of transgenic plants; knockout of ORF1 in wild-type plants by Crispr technology resulted in a decrease of seed setting rate in all eight positive families. 2. ORF1 encodes a cyclic nucleotide-gated ion channel (cyclic cn-n). Ucleotide-gated channels (CNGCs) protein OsCNGC13, which controls spikelet fertility by regulating programmed cell death in the style, was found to be a constitutive expression pattern and accumulated in the pistil. The rice and tobacco systems indicated that the subcellular localization of OsCNGC13 was in the cell membrane. GC13 is a calcium channel protein. There are three calmodulin in rice and all of them can interact with OsCNGC13, but no homologous protein can interact with OsCNGC13 in 16 members of the rice CNGC family. In addition, the mutant was found to be lighter and less extensive than the wild type. At 30 minutes after anthesis, dead cells in the pollen tube passage of 9311 were observed, but the corresponding parts of the mutant were still intact. A large number of intercellular spaces were observed in the 9311 pollen tube channel, but only a few cells died out in the mutant. 3. OsZHHD1 controlled rice leaf curl. Cytological observation of the anti-curling mutant showed that the number of vesicular cells in the mutant leaves increased and arranged abnormally. Overexpression of the homeobox transcription factor OsZHD1 results in leaf roll reversal in rice. OsZHD1 is constitutively expressed and accumulated in developing leaves and spikes. Overexpression of OsZHHD1 in wild type results in leaf roll reversal in all transgenic positive families.
【学位授予单位】：南京农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S511

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