水稻叶片表皮蜡质合成基因DRP3克隆与功能验证

Plant epidermal waxes serve as a protective barrier against water loss, UV light, pathogens and insects. The epidermal waxes are complex mixtures of C20–C34 very-long-chain fatty acids (VLCFAs) and their derivatives including aldehydes, primary alcohols, secondary alcohols, alkanes and esters. The waxes are synthesized and secreted by epidermal cells. To date, the researches about biosynthesis, transportation and deposition of epidermal waxes are mainly restricted in Arabidopsis, less in rice. We have obtained a rice dripping wet leaf (DRP) mutant, drp3, which lacked epidermal wax in leaves by fertilized egg treatment with MNU. drp3 mutant differed from the rice wax sparse leaf mutnats reported by that its alkane content increased dramatically. We have cloned the DRP3 gene via map-based cloning strategy and verified the gene through phenotype rescure experiment. DRP3 gene encodes a member of cytochrome CYP96B subfamily. Based on these results, we will characterize the functions of DRP3 gene via reverse genetical, biochemical, molecular and cellular biological strategies. We also hope to identify the substrates and products of the putative CYP96B enzyme. The expected results will help us to reveal the molecular mechanisms concerning epidermal wax synthesis, transportation and deposition. The DRP3 gene being determined will contribute greatly to the researches of epidermal wax synthesis, intracellular transportation and deposition and development of new crop cultivars with high tolence/resistance to bio- and abiostress by biotechnological strategies.

植物叶表面蜡质对于减少水分蒸腾、提高耐旱性、减弱紫外光伤害以及抵抗病虫害具有重要作用。目前对植物叶表面蜡质合成代谢机理的研究主要限于拟南芥中，对水稻叶表面蜡质合成代谢途径的报导不多。利用化学诱变剂MNU处理受精卵方法，我们获得了一个水稻叶表面蜡质缺失突变体(drp3)。与已报导的水稻蜡质缺失突变体不同，drp3突变体叶表皮蜡质中烷烃含量极显著增加。利用图位克隆方法已分离到DRP3基因，并通过表型回复实验确认。该基因编码一个功能未知的细胞色素P450家族 CYP96B亚家族蛋白。本研究将在现有研究基础上，利用反向遗传学、分子生物学、细胞生物学、生物化学等手段，对其功能进行详尽研究，确定CYP96B亚家族蛋白的作用底物及产物，以揭示DRP3基因在叶表皮蜡质合成代谢中的作用。对于揭示叶表面蜡质合成代谢调控机制，具有重大的理论意义。同时对于利用生物技术手段培育抗逆作物新品种，具有重要的实践意义。

植物表皮蜡质对于减少水分蒸腾、提高耐旱性、减弱紫外光伤害以及抵抗病虫害等具有重要作用。蜡质主要由超长链脂肪酸及其衍生物（醛、醇、烷烃、酮和酯类等）组成。超长链脂肪酸分别进入酰基还原途径生成偶数碳链的一级醇和酯类和脱羰途径生成偶数碳链的醛和奇数碳链的烷烃。在拟南芥茎表皮中烷烃进一步转化为二级醇和酮，而水稻等单子叶植物叶表皮蜡质中却不含二级醇和酮。因此，烷烃被认为是单子叶植物脱羰途径的最终产物。与此同时，奇数碳链一级醇是蜡质的组成成份，但其合成途径尚不清晰。.本研究从水稻MNU诱变突变体库中筛选获得一个叶片呈沾水表型的dripping-wet leaf 3 (drp3)突变体。突变体叶片表皮蜡质中烷烃异常累积，而C29一级醇含量极显著降低。通过图位克隆的方法，分离出控制该性状的DRP3基因，编码一个功能未知的细胞色素P450家族蛋白CYP96B5。研究发现, CYP96B5定位于内质网，其过量表达转基因水稻叶表皮蜡质中烷烃含量极显著降低，C29一级醇含量增加；而抑制表达突变体则呈相反表型。异源表达的DRP3/CYP96B5与拟南芥内源烷烃链中羟化酶MAH1/CYP96A15共同竞争烷烃底物，导致前者产物C29一级醇含量增加，后者产物二级醇和酮含量降低。DRP3与饱和烷烃共同注射烟草叶片试验进一步证明其功能。这些结果表明，DRP3/CYP96B5以烷烃为底物催化产生一级醇。该研究首次在植物中发现烷烃末端羟化酶，阐明植物中超长奇数碳链伯醇合成的机理。