玉米hzms1 突变体杂合雄性不育的分子机理研究

Meiosis is a key process during plant gametes development, and is precisely regulated by a series genes. In this project, we study a new type of maize male sterile mutant hzms1, the heterozygous plants showed pollen sterile causing by the highly chromosome condensation at pachytene stage and the following processes, while the homozygous mutants had no obvious defects. By using map based cloning strategy and transgenic method, we confirmed that a piont mutation in a plant invertase coding gene causing the defects of hzms1 heterozygous plant. In this project, we plan to perform the following research: analyzing the hzMS1 gene function with hzMS1-knockout mutants generated by CRISPR/Cas9 system, confirming the detailed gene temporal and spatial expression pattern with mRNA in situ hybridization assay, measuring the sugar content with metabolism analysis, detecting the enzyme features of hzMS1 protein with prokaryotic expression system and enzyme activity assay, analyzing gene differential expression via RNA sequencing. There are two main goals of this project, firstly, deciphering the mechanism of heterozygous male sterile phenotype of hzms1, secondly, clarifying the molecular function of hzMS1 in maize microspore meiosis process. This study would expand our knowledge about meiosis regulation, and will provide the gene resource and theoretical basis for new maize male sterile line development.

减数分裂是植物育性形成过程中的一个关键环节，受到一系列基因的精密调控。本项目研究对象是一个新型玉米雄性不育突变体hzms1, 杂合突变体从减数分裂粗线期开始，染色体高度浓缩，花粉败育，而纯合突变体育性基本正常。申请者前期通过基因图位克隆的策略和转基因验证,确定了一个植物转化酶基因为目的基因。本研究拟应用CRISPR/Cas9 技术进一步对hzMS1 基因进行功能分析，采用mRNA原位杂交深入研究基因的时空表达模式，以蛋白质免疫组化确定蛋白富集的模式，利用代谢组分析研究突变体糖分等物质含量的变化，再以原核表达和酶活性分析确定hzMS1 蛋白的酶学性质，采用转录组测序的方法研究基因表达的变化。通过本项目的研究，解析hzms1杂合突变体雄性不育的机制，阐明hzMS1 基因参与玉米花粉母细胞减数分裂调控的分子机理，为开发新型雄性不育系提供基因资源和理论支持。

对有性生殖的植物而言，减数分裂过程的正常进行是产生可育配子的前提条件。在该研究中，通过克隆玉米的一个显性突变体hzMs1/Mei025，发现一个新的细胞质转化酶参与减数分裂过程。Mei025/+杂合突变体花粉母细胞粗线期之前的减数分裂过程没有明显异常，而在粗线期之后，染色体高度浓缩，减数分裂停滞。通过基因图位克隆和转基因验证，发现Mei025的突变表型是由于一个细胞质转化酶编码基因INVERTASE ALKALINE NEUTRAL 6 (INVAN6)第二外显子上的一个点突变造成的。在Mei025 突变体中，INVAN6的第276位天冬氨酸被替换成为天冬酰胺。INVAN6主要在花粉母细胞中表达。与杂合突变体几乎完全雄性不育不同，Mei025 纯合突变体部分花粉母细胞的减数分裂过程正常，最终表现为雄性半不育。根据遗传学证据，推测野生型INVAN6和突变型INVAN6Mei025共存时对花粉母细胞减数分裂过程有抑制作用。INVAN6特异水解蔗糖，而突变型的INVAN6Mei025不具有转化酶活性。糖分含量和转录组分析表明，INVAN6Mei025存在会影响花药中糖分积累、转运和糖信号。通过该研究，为解析糖代谢或糖信号基因参与减数分裂调控提供新的线索。