拟南芥固醇酮基还原酶(SKR)编码基因的鉴定及其功能的研究

Sterols become functional only after removal of the two methyl groups at C-4. Each C-4 demethylation step involves the sequential participation of three individual enzymatic reactions including a sterol 4α-methyl oxidase (SMO), a bifunctional 4α-carboxysterol-C3-dehydrogenase/C4-decarboxylase (CSD), and a sterone ketoreductase (SKR). In Arabidopsis, two families of SMO (SMO1 and SMO2, containing three and two members, respectively) acting on the first and the second C-4 demethylation step, respectively. There are also two CSD enzymes in Arabidopsis, which are not as specific as the SMOs for a particular substrate, so they can perform in both C-4 demethylation steps. However, the number of SKR in Arabidopsis, and its substrates specification remain unknown. SKR was demonstrated to be essential for yeast and mammal survival. SKR deficient mouse was embryo lethal, and died at early embryo developmental stage. However, the role of SKR in plant development is still unknown. To identify the Arabidopsis SKR encoding gene, we had investigated the Arabidopsis genome using bioinformatics approach, and had found four potential candidates for SKR encoding sequences. We had isolated mutants for these genes, and found that one of the mutants was dwarf, bushy, and sterile, which was similar to sterol biosynthetic mutants such as smt1, fackel and hydra1. We had determined the subcellular localization of these putative SKR proteins, and found that they all localized to the endoplasmic reticulum, the location for sterol biosynthesis. These results suggested that these candidates might be the SKR genes of Arabidopsis. We will further analyze these candidates’ ability to complement SKR-deficient yeast and to reduce the 3-ketosteroids. Our study will identify the elusive SKR in Arabidopsis, and reveal its function in higher plant development.

固醇生物合成过程中必须去除C-4位的两个甲基才能成为有功能的固醇，C4-甲基的去除是由固醇4α-甲基氧化酶（SMO）、4α-羧基固醇-C3-脱氢酶/C4-脱羧酶(CSD)和固醇酮基还原酶(SKR)依次反应完成的。已知拟南芥中有5个SMO和2个CSD同工酶，但是SKR的数目和底物特异性尚未有任何研究报道。酵母和哺乳动物中SKR缺失都是致死的，而SKR在植物生长发育中的功能完全未知。我们通过生物信息学分析获得了4个拟南芥SKR候选基因，其中一个基因的突变体植株矮化、丛生、不育，这与已报道的固醇突变体的表型相似，而且亚细胞定位结果显示这4个基因编码的蛋白定位于内质网（固醇合成场所）。这些结果预示它们是SKR基因。本课题拟进一步通过遗传学和生物化学等手段对这些基因进行鉴定，并阐明SKR的底物特异性和生物学功能，从而完善我们对于植物固醇合成途径的了解，为进一步研究植物固醇合成的分子调控机制奠定基础。

固醇生物合成过程中必须去除C-4位的两个甲基才能成为有功能的固醇，C4-甲基的去除是由固醇4α-甲基氧化酶（SMO/ERG25）、4α-羧基固醇-C3-脱氢酶/C4-脱羧酶(CSD/ERG26)和固醇酮基还原酶(SKR/ERG27)依次反应完成的，这些酶通过脚手架蛋白ERG28形成一个固醇C-4脱甲基多酶复合体（SC4DM）。酵母和哺乳动物中的SKR/ERG27的编码基因已经被鉴定。虽然植物中已经证明具有SKR的酶活性，但是其编码基因尚未被鉴定。已知拟南芥中有两个底物特异性不同的SMO亚家族（SMO1和SMO2，分别包含3个和2个成员）、2个CSD同工酶和一个AtERG28蛋白，但是SKR的数目和底物特异性完全未知。另外，是否拟南芥中相应的存在两个SC4DM复合体还有待研究，而SMO1亚家族的生物学功能也未被阐明。本项目研究通过酵母双杂交筛库和回补酵母erg27突变体鉴定到了拟南芥中可能的AtSKR基因，并且发现拟南芥中只有一个同源基因。序列比对和进化树分析表明该基因在植物中高度保守。AtSKR基因在拟南芥的多数组织均有表达，在幼嫩的角果中表达水平最高。atskr的突变体表现出矮化的表型，其表型与固醇合成途径下游的基因例如DWARF7、DWARF5和DWARF1的突变体的表型相似，这暗示调控胚发育的固醇分子可能是SKR上游的固醇中间产物。通过对拟南芥SC4DM复合体蛋白之间的互作研究，发现可能存在两类SC4DM复合体，一类包含SMO1亚家族蛋白，一类包含SMO2亚家族蛋白，而且SMO2亚家族蛋白不与CSD1和CSD2互作，而是与CSD3和CSD4（我们新鉴定到的CSD）相互作用。另外，我们发现SMO1基因亚家族在胚发育过程中有重要的功能，其通过维持正常的固醇成分而影响胚发育过程中生长素和细胞分裂素的活性，从而影响胚的发育。

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