‘741杨’D2类细胞周期蛋白基因的克隆及功能鉴定  

Cloning and Function Identification of D2-Type Cyclin Gene in‘741 Poplar’

作　　者：刘孝晨 关超男 许楠 诸葛祥林 杨海灵[1] LIU Xiaochen;GUAN Chaonan;XU Nan;ZHUGE Xianglin;YANG Hailing(College of Biological Sciences and Biotechnology, Beijing Forestry University, Beijing 100083, China)

机构地区：[1]北京林业大学生物科学与技术学院,北京100083

出　　处：《西北植物学报》2020年第4期547-556,共10页Acta Botanica Boreali-Occidentalia Sinica

基　　金：国家自然科学基金(31570664)。

摘　　要：D类细胞周期蛋白(D-type cyclin,CYCD)调控细胞周期G1/S期转变。CYCD与细胞周期蛋白依赖性激酶(cyclin-dependent kinase,CDK)结合形成CYCD/CDK复合物,被激活的CYCD/CDK复合物通过磷酸化下游细胞周期响应因子调控细胞周期有序进行,进而影响植物的生长发育。该研究以‘741杨’为实验材料,成功鉴定得到1个D2类细胞周期蛋白基因(PtoCYCD2;1)。研究表明:(1)实时定量PCR(qRT-PCR)显示,PtoCYCD2;1基因在根、茎、叶、叶柄、树皮和木质部中均有表达,在叶中的相对表达水平最高。(2)亚细胞定位表明PtoCYCD2;1蛋白定位于细胞核。(3)与野生型(Wild-Type poplar,WT)相比,过表达PtoCYCD2;1基因的‘741杨’出现株高降低,茎直径减小,叶片明显下卷的表型。(4)扫描电镜分析(SEM)显示,转基因杨树叶片的上表皮细胞平均面积变小,细胞数量增多;树脂切片结果显示与WT相比,转基因杨树叶片的栅栏组织和海绵组织的细胞间隙疏松。(5)qRT-PCR结果显示,转基因杨树中细胞周期调控基因CDKA;1、CDKB1;1和CDKB2;1的表达水平显著上调,植物成视网膜细胞瘤相关蛋白1(retinoblastoma-related protein1,RBR1)基因、细胞周期蛋白依赖性激酶抑制因子(kip-related protein,KRP)基因的表达水平显著下调。该研究结果为进一步研究木本植物CYCD2基因的功能奠定了基础。D-type cyclin(CYCD)regulates the G1/S phase transition of the cell cycle.CYCD combines with Cyclin-Dependent Kinase(CDK)to form CYCD/CDK complex.The activated CYCD/CDK complex regulates the cell cycle through phosphorylation of downstream cell cycle response factors,which in turn affects plant growth and development.In this study,‘741 poplar’was used as experimental material,and a D2-type cyclin gene was identified(PtoCYCD2;1).The results showed that:(1)Quantitative real time polymerase chain reaction(qRT-PCR)showed that PtoCYCD2;1 gene was expressed in roots,stems,leaves,petioles,bark,and xylem,with the highest relative expression in leaves.(2)Subcellular localization indicated that PtoCYCD2;1 protein was localized in the nucleus.(3)Compared with the wild-type poplar(WT),‘741 poplar’overexpressing PtoCYCD2;1 gene showed reduced plant height,reduced stem diameter,and significantly lower leaf volume.(4)Scanning Electron Microscope(SEM)analysis showed that cells of transgenic poplars were smaller and more numerous than those of WT.Resin section showed that,compared with WT,the intercellular space of the fence tissue and sponge tissue of the transgenic poplar leaves was loose.(5)qRT-PCR showed that,overexpressing PtoCYCD2;1 gene in‘741 poplar’,the expression levels of CDKA;1,CDKB1;1 and CDKB2;1 were significantly up-regulated,while retinoblastoma-related protein1(RBR1)gene,cyclin-dependent kip-related protein(KRP)gene expression level were significantly down-regulated.This study lays a foundation for further understanding the function of CYCD2 gene in woody plants.

关 键 词：‘741杨’ 细胞周期蛋白 PtoCYCD2 1基因 基因表达 亚细胞定位 功能分析 

分 类 号：Q785[生物学—分子生物学] Q786