水稻OsPP1a基因克隆和RNAi-OsPP1a遗传转化分析

Cloning of OsPP1a from rice and analysis of its RNAi-OsPP1a genetic transformation

为了研究水稻中蛋白磷酸酶PP1的功能,本文以拟南芥中AtPP1a蛋白序列为检索序列,在水稻基因组中搜索获得其高度同源基因序列,基因号为LOC_Os03g16110,设计引物在水稻‘中花11’中克隆该基因并命名为OsPP1a。系统进化分析表明,拟南芥PP1a与水稻PP1a亲缘关系最近,同源性高达90%。构建了RNAi-OsPP1a载体,并成功转化‘中花11’受体愈伤组织,筛选得到了15株转基因阳性植株,其中有4株表型矮化,实时荧光定量PCR(qRT-PCR)检测叶片组织中OsPP1a表达量极显著降低,说明RNAi-OsPP1a能够显著降低株高。构建过表达OsPP1a cDNA载体并转化‘中花11’,获得12株转基因阳性植株,5株成熟植株的株高显著性增加,叶片组织中OsPP1a表达量显著增加。研究结果表明OsPP1a在调控水稻株高中发挥重要作用,功能比较保守。

In order to study the function of protein phosphatase PP1 in rice(Oryza sativa),the Arabidopsis thaliana PP1 a protein sequence was used as a query to search the rice genome.We obtained its homologous gene sequence,whose genetic code was LOC_Os03 g16110,and the target gene was cloned from‘Zhonghua11’and it was named OsPP1 a.Phylogenetic analysis showed that the AtPP1 a had the closest relationship with the OsPP1 a,with homology up to 90%.RNAi-OsPP1 a vector was constructed and successfully transformed into callus of‘Zhonghua 11’receptor,and 11 transgenic positive strains were screened,among which four plants showed phenotypic dwarfism.RNAi-OsPP1 a significantly decreased the expression of OsPP1 a by Real-time quantitative PCR(qRT-PCR),indicating that RNAi-OsPP1 a could significantly reduce plant height.Overexpressed vector was constructed and 9 transgenic positive plants were obtained.Overexpression of OsPP1 a(OE-OsPP1 a)cDNA in‘Zhonghua 11’caused a significant increased plant height in 5 transgenic lines,while the others showed no change.The expression of OsPP1 a in OE-OsPP1 a transgenic lines were significantly increased.These results showed that OsPP1 a plays an important role in regulating rice plant high and its function was relatively conservative with Arabidopsis.