OsRhoGDI2过表达转基因水稻的筛选鉴定及外源基因拷贝数的初步分析
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摘要
水稻Rho GDP解离抑制基因OsRhoGDI2是从幼穗中分离出的功能未知基因。为鉴定该基因的功能,笔者所在实验室前期构建了植物过表达载体pCAMBIA1302-OsRhoGDI2-GFP,并对水稻进行了遗传转化。对OsRhoGDI2过表达转基因水稻T_2代进行筛选和鉴定,采用PCR技术鉴定转基因植株,采用半定量RT-PCR和实时荧光定量PCR检测OsRhoGDI2在转基因水稻中的表达水平,结果显示,其中6个株系为过表达转基因植株,OsRhoGDI2表达水平上调1.69～13.35倍。为检测外源基因在转基因水稻中的拷贝数,分别以蔗糖磷酸合成酶基因SPS和潮霉素抗性基因HYG为内参基因和标记基因,采用实时荧光定量PCR(qPCR)技术结合内参基因和标记基因的标准曲线进行分析,结果显示在所检测的6个转基因株系中,外源基因的拷贝数均为1,提示已经获得稳定遗传的OsRhoGDI2过表达转基因水稻,为后续OsRhoGDI2基因的功能研究奠定基础。
引文
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[2]Molendijk A J,Bischoff F,Rajendrakumar C S,et al.Arabidopsis thaliana Rop GTPases are localized to tips of root hairs and control polar growth[J].EMBO Journal,2001,20(11):2779-2788.
[3]Jones M A,Shen J J,Fu Y,et al.The Arabidopsis Rop2 GTPase is a positive regulator of both root hair initiation and tip growth[J].The Plant Cell,2002,14(4):763-776.
[4]Fu Y,Wu G,Yang Z B.Rop GTPase-dependent dynamics of tiplocalized F-actin controls tip growth in pollen tubes[J].Journal of Cell Biology,2001,152(5):1019-1032.
[5]Fu Y,Li H,Yang Z B.The ROP2 GTPase controls the formation of cortical fine F-actin and the early phase of directional cell expansion during Arabidopsis organogenesis[J].The Plant Cell,2002,14(4):777-794.
[6]Delmer D P,Pear J R,Andrawis A,et al.Genes encoding small GTP-binding proteins analogous to mammalian rac are preferentially expressed in developing cotton fibers[J].Molecular&General Genetics,1995,248(1):43-51.
[7]Trotochaud A E,Hao T,Wu G,et al.The CLAVATA1 receptor-like kinase requires CLAVATA3 for its assembly into a signaling complex that includes KAPP and a Rho-related protein[J].The Plant Cell,1999,11(3):393-406.
[8]Kawasaki T,Henmi K,Ono E,et al.The small GTP-binding protein rac is a regulator of cell death in plants[J].Proceedings of the National Academy of Sciences of the United States of America,1999,96(19):10922-10926.
[9]Kawasaki T,Koita H,Nakatsubo T,et al.Cinnamoyl-CoAreductase,a key enzyme in lignin biosynthesis,is an effector of small GTPase Rac in defense signaling in rice[J].Proceedings of the National Academy of Sciences of the United States of America,2006,103(1):230-235.
[10]Fujiwara M,Umemura K,Kawasaki T,et al.Proteomics of Rac GTPase signaling reveals its predominant role in elicitor-induced defense response of cultured rice cells[J].Plant Physiology,2006,140(2):734-745.
[11]Schultheiss H,Hensel G,Imani J,et al.Ectopic expression of constitutively activated RACB in barley enhances susceptibility to powdery mildew and abiotic stress[J].Plant Physiology,2005,139(1):353-362.
[12]Dermardirossian C,Bokoch G M.GDIs:central regulatory molecules in Rho GTPase activation[J].Trends in Cell Biology,2005,15(7):356-363.
[13]Dovas A,Couchman J R.RhoGDI:multiple functions in the regulation of Rho family GTPase activities[J].The Biochemical Journal,2005,390:1-9.
[14]Klahre U,Becker C,Schmitt A C,et al.Nt-RhoGDI2 regulates Rac/Rop signaling and polar cell growth in tobacco pollen tubes[J].Plant Journal,2006,46(6):1018-1031.
[15]Bischoff F,Vahlkamp L,Molendijk A,et al.Localization of AtROP4and AtROP6 and interaction with the guanine nucleotide dissociation inhibitor AtRhoGDI1 from Arabidopsis[J].Plant Molecular Biology,2000,42(3):515-530.
[16]Kieffer F,Elmayan T,Rubier S,et al.Cloning of Rac and RhoGDI from tobacco using an heterologous two-hybrid screen[J].Biochimie,2000,82(12):1099-1105.
[17]梁卫红,唐朝荣,吴乃虎.两种水稻GDP解离抑制蛋白基因的分离及特征分析[J].中国生物化学与分子生物学报,2004,20(6):785-791.
[18]Carol R J,Takeda S,Linstead P,et al.A RhoGDP dissociation inhibitor spatially regulates growth in root hair cells[J].Nature,2005,438(770):1013-1016.
[19]Wu Y X,Zhao S J,Tian H,et al.CPK3-phosphorylated RhoGDI1is essential in the development of Arabidopsis seedlings and leaf epidermal cells[J].Journal of Experimental Botany,2013,64(11):3327-3338.
[20]彭威风,梁卫红.水稻OsRho GDI2蛋白生物信息学分析及亚细胞定位研究[J].生物技术通报,2010(5):82-86,92.
[21]Huang J J,Zhang J,Hao Y F,et al.Distinct expression patterns of the GDP dissociation inhibitor protein gene(OsRhoGDI2)from Oryza sativa during development and abiotic stresses[J].Biologia,2016,71(11):1230-1239.
[22]Livak K J,Schmittgen T D.Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCTmethod[J].Methods,2001,25(4):402-408.
[23]杨立桃,赵志辉,丁嘉羽,等.利用实时荧光定量PCR方法分析转基因水稻外源基因拷贝数[J].中国食品卫生杂志,2005,17(2):140-144.
[24]Ding J Y,Jia J W,Yang L T,et al.Validation of a rice specific gene,sucrose phosphate synthase,used as the endogenous reference gene for qualitative and real-time quantitative PCR detection of transgenes[J].Journal of Agricultural&Food Chemistry,2004,52(11):3372-3377.
[25]Flavell R B.Inactivation of gene expression in plants as a consequence of specific sequence duplication[J].Proceedings of the National Academy of Sciences of the United States of America,1994,91(9):3490-3496.
[26]Vaucheret H,Béclin C,Elmayan T,et al.Transgene-induced gene silencing in plants[J].The Plant Journal,1998,16(6):651-659.