水稻LHD基因调节抽穗期的分子机理研究

The flowering time, often referred as heading date in cereal crops, is considered as one of the critical traits for adapting rice to different cultivation areas and cropping seasons. The genes and molecular mechanisms controlling flowering time have been extensively studied in the model dicot Arabidopsis. Comparative studies between Arabidopsis and rice have shown that rice has homologues of many flowering-time genes and the genes involved in the photoperiod pathway are well conserved between the two species. Distinct effects of some flowering regulators were, however, also observed in rice alone, implying that different mechanism for flowering controls may exist in cereal crops. To understand the molecular mechanisms underlying heading, the rice mutant lhd (late heading date) was identified in M2 generation from the mutant library of rice (Oryza sativa cultivar Zhonghua 11) induced by gamma rays (Co60). The lhd mutant was flowered about 45 days later than the wild type (WT) under the natural condition in fall (autumn) of Hangzhou and spring of Lingshui, Hainan, respectively. Meanwhile, the lhd showed more tilling and internode number, and smaller grain than that of the wild type. Segregation analysis indicated that lhd was a recessive mutation. The mutant gene LHD identified from map-based cloning is a new locus involved in regulating heading date in rice, encoding a MYB family transcription factor. The mutant phenotypes were rescued by transformation with a construct containing full-length LHD genomic DNA, confirming that this locus is responsible for the mutant phenotype. To test whether LHD gene involving the circadian rhythm and photoperiod flowering pathway, the real-time PCR was performed to analysis some genes known to affect heading date, such as Hd1, Ehd1 and Hd3a etc, under short day conditions (SD), the diurnal expression profiles and peak levels of these genes were altered in lhd, suggesting that LHD was probably involve in regulated circadian rhythms and photoperiod flowering pathway in rice. This study is aiming to elucidate the biological function of LHD and the molecular mechanisms underlying the circadian clock and photoperiodic control of flowering time in rice. The outcomes from this study will hopefully provide the theoretical basis for rice adapting to different environments and regions, and finally help for rice domestic and variety improvement.

水稻开花（抽穗）时间决定了水稻品种的季节和地区适应性,与产量和品质密切相关。本实验室从Co60辐射处理的中花11突变体库M2代中筛选到一个晚抽穗的突变体lhd (late heading date)，在杭州夏季和海南陵水春季自然条件下，lhd比野生型中花11的抽穗期均晚45天以上，同时，lhd的分蘖数目和节间数量有所增加，籽粒变小。遗传学实验证实lhd受一对隐性核基因控制，随后通过图位克隆法分离到的LHD基因编码MYB家族的一个转录因子，是控制水稻抽穗期的新基因位点。功能互补实验已证实LHD是引起lhd突变的基因，通过定量PCR分析发现多个控制抽穗期的基因在lhd中的表达节律和表达峰发生了改变，提示LHD基因可能参与生物节律和光周期信号调节。本研究拟对LHD基因进行深入的功能研究，阐明LHD基因参与水稻抽穗期调控的分子机理，为水稻的广泛引种和改良水稻新品种的地区适应性提供理论依据。

作物开花时间决定作物的产量，品质和地区适应性。 因此，对水稻的开花研究成为重要的研究课题。在克隆水稻LHD基因的基础上，对lhd突变体进行了系统的表型鉴定和功能分析。lhd无论长日照还是短日照，抽穗期均比对照晚45天左右，表明lhd突变体不受光周期影响。对lhd进行农艺性状鉴定，发现lhd的分蘖数目和节间数量有所显著增加，籽粒变小，结实率下降。LHD基因编码一个转录因子，是控制水稻抽穗期的新基因位点。LHD蛋白定位于细胞核中，LHD基因主要在叶片中表达，利用BiFC分析了LHD与ELF3的互作，发现LHD与ELF3可能以复合体的形式存在。通过定量PCR分析发现，LHD基因主要通过抑制PRRs基因表达，促进OsLHY1基因表达参与生物钟循环，此外，通过对开花基因的表达分析发现，LHD主要通过促进抑制Ghd7和促进Ehd2/Rid1的表达发挥作用，提示LHD主要通过Ehd1途径发挥作用。利用RiceVarMap网络数据库发现LHD基因在进化中受到了自然选择或人工选择。本研究为水稻的广泛引种和改良水稻新品种的地区适应性提供了理论依据，也为水稻的驯化和改良提供了支持。