水稻光叶突变新基因的克隆和亚细胞定位  

Cloning and Subcellular Localization of a New Glabrous-Leaf Mutant Gene GLL in Rice(Oryzα sativα L.)

作　　者：董陈文华 张小玲[1] 朱骞[1] 熊海波[1] 魏振飞[2] 吕永刚[3] 张利东[1] 伍腾飞[1] 李伟[1] 吴超[1] 陈丽娟[1] 李东宣[4] 

机构地区：[1]云南农业大学稻作研究所,昆明650201 [2]山西省农业科学院玉米研究所,忻州034000 [3]云南省农业科学院粮食与作物研究所,昆明650205 [4]云南省高校滇型杂交粳稻分子育种重点实验室,昆明650201

出　　处：《分子植物育种》2015年第4期716-726,共11页Molecular Plant Breeding

基　　金：NSFC-云南联合基金重点项目(U1136604);国家重点基础研究973项目(2011CB100401);云南省水稻产业技术体系(A3006516)共同资助

摘　　要：水稻叶片表皮毛突变体是研究单子叶植物细胞分化和水稻品种改良的重要材料。本研究对T-DNA标签技术获得的水稻光叶突变体(单隐性核基因控制,稳定遗传)进行田间观测和扫描电镜观察,以分析突变体叶片毛状体类型和分布特征;并采用DNA-Walking和RT-PCR方法定位并克隆该突变体基因,通过对该基因进行初步的生物信息学分析,构建绿色荧光蛋白融合载体,进行亚细胞定位。研究结果显示:突变体叶缘无毛,叶面毛状体缺失,缺失的毛状体类型主要为宏毛(即钩毛),但种子颖壳表面毛状体正常生长,是国内外尚未报道的一种新型的光叶突变体;对T-DNA标签插入序列进行相似性比对,确定插入位点,表明突变体候选基因在水稻第6号染色体上,通过基因克隆获得了c DNA序列,暂命名为GLL;生物信息学分析表明,GLL基因含有8个外显子,7个内含子,该基因的开放阅读框(ORF)有702 bp,编码233个氨基酸;对GLL蛋白结构域进行分析,发现该蛋白有一个PEX11保守结构域;系统进化关系表明,GLL与预测的水稻中PEX11-5同源性最高;亚细胞定位表明该基因在细胞核、细胞质和细胞膜中表达。本研究为解析水稻及单子叶植物中毛状体发育分子机制和进行水稻品种改良奠定了基础。Rice leaf trichome mutant is an important germplasm for study on monocot cell differentiation and rice breed improvement. In this study, the glabrous leaf mutant was generated by T-DNA insert method, which was governed by a single recessive nuclear gene with stable hereditary. In this research we did the observation of the trichome on the leaf and hull by SEM （scanning electron microscope）, and mapped and cloned the tentative gene through the approaches of DNA-Walking and RT-PCR, and analyzed the sequence of the gene as well, furthermore we built the fusion vector of the gene that was fused with enhance green fluorescent protein for subcellular localization. The results revealed that there was no hair on the edge of leaf, and the trichomes were normal on the hull but missed on the leaf surface, the missed trichomes were bristle type macrohair, therefore we realized this mutant might be a novel glabrous mutant in rice. Based on T-DNA insertion tag and the insert-sequence position analysis, the candidate gene was located on chromosome 6 in rice and the cDNA sequence was cloned, which was tentatively named GLL. Bioinformatics analysis showed that GLL contained 8 exons and 7 introns; the open reading frame （ORF） of GLL was 702 bp, encoded 233 amino acid residues. The structure analysis revealed that GLL protein had a conserved PEX11 domain. Phylogenetic analysis showed that GLL shared the highest similarity with the PEX11-5 protein in rice. The subcellular localization showed that the expression of GLL gene and EGFP fusion protein was mainly localized in the nucleus of onion epidermal cell, and also expressed in cytoplasm and cell membrane. This study provided molecular basis of the trichome development in rice and monocot plant for futher exploitation on rice breed improvement.

关 键 词：水稻 光叶突变体 GLL基因 亚细胞定位 序列分析 

分 类 号：Q943.2[生物学—植物学]