柑橘雄性不育类蛋白基因MSLP的克隆与分析

Cloning and Functional Analysis of Male Sterile 2-like Protein Gene(MSLP)in Citrus

为分析柑橘雄性不育类蛋白基因MSLP的功能,利用RT-PCR法在6个典型柑橘品种中克隆了MSLP cDNA全长,检测到4种不同的转录本,其中MSLP-B型(1476 bp)在6个品种中均能克隆到。序列和系统进化分析表明,MSLP包含3个脂酰辅酶A还原酶(fatty acyl-CoA reductase,FAR)C端结构域,MSLP-B与甜橙FAR1-like蛋白和克里曼丁橘FAR3蛋白聚在一类,与蔷薇科植物如碧桃、月季、甜樱桃和梅的FAR蛋白亲缘关系最近;MSLP在柑橘基因组有5~8个拷贝数,MSLP-B作用于细胞质,在无核椪柑各时期花器官中的表达量均高于普通有核椪柑;MSLP-B转基因烟草表型分析表明,转基因植株成熟期花柱/花丝比值高于野生型,尽管花粉活力不受影响,但不饱满的椭圆形花粉粒显著增多。

To elucidate the functions of male sterile 2-like protein(MSLP)in citrus,the full-length cDNAs of MSLP were cloned in six typical citrus varieties using RT-PCR.Four transcripts of MSLP were identified,and only MSLP-B existed in all varieties.Structure analysis showed MSLP contained three conserved fatty acyl-CoA reductase(FAR)C terminal domains.Phylogenetic analysis suggested that MSLP-B clustered with sweet orange FAR-like protein and clementine FAR3 protein,and they had closer genetic relationship with FAR-proteins in rosaceous plant like peach,rose,sweet cherry and plum.Southern blotting showed there were 5-8 copies of MSLP in citrus.Transient expression of MSLP-B-GFP in Nicotiana benthamiana leaves indicated it functioned in cytoplasm.qRT-PCR results showed the expression of MSLP-B in floral organs of seedless Ponkan was up-regulated compared with common seedy Ponkan.Over-expression of MSLP-B in tobacco showed that the ratio of stylar length to filament length in mature flower was higher than non-transgenic tobacco.The viability of pollen grains was normal in transgenic lines.However,the number of unsaturated elliptic pollen grains increased significantly.