摘要
类胡萝卜素裂解双加氧酶7 (CCD7)、类胡萝卜素裂解双加氧酶8 (CCD8)基因对于植物的侧枝及侧根生长有着重要的影响。本研究以沙柳为研究材料,克隆获得CCD7、CCD8基因序列,进行了生物信息学分析、组织表达特异性及预测分析,为沙柳CCD7、CCD8基因的功能研究及利用奠定基础,丰富了木本植物中CCD基因家族的研究。根据拟南芥、黄瓜CCD7、CCD8同源基因序列,由数据库Phytozome 11获得红皮柳、毛果杨的同源序列,由沙柳中进行同源基因克隆。利用ExPASy数据库预测蛋白理化特性、氨基酸组成。利用cNLS mapper在线预测蛋白核定位信号(NLS)。采用T-COFFEE和MEGA 6.0比对SpsCCD7、SpsCCD8氨基酸序列同源性和构建系统发育树。克隆获得的CDS长分别为1 843、1 674 bp,由614、557个氨基酸残基构成,SpsCCD7、Sps CCD8蛋白无跨膜结构和信号肽区域,其理论等电点(pI)及分子量分别为7.55、6.10,69.299 5、61.981 5 kD,认定均为亲水性蛋白。系统进化树及氨基酸序列比对分析表明,SpsCCD7、SpsCCD8与毛果杨和红皮柳亲缘关系最高,且SpsCCD7、SpsCCD8与毛果杨、红皮柳、水稻等植物的CCD7、CCD8基因之间具有三个RPE65基因家族的典型保守区域Motif region,其隶属于CCD蛋白家族,但在序列末端也存在一定差异。在定量分析中,沙柳SpsCCD7、SpsCCD8基因在各组织(根,花,叶,芽,茎尖,嫩茎)均有表达,SpsCCD7基因在茎尖中表达量最高,SpsCCD8基因在根中表达量最高。其组织特异性表达与其他木本植物存在一定差异,这种表达差异可能与沙柳本身为灌木木本植物相关。本研究克隆获得了沙柳CCD7、CCD8基因,隶属CCD基因家族,为进一步探索木本植物CCD基因家族对分枝的调控机理奠定了基础,为未来培育丛生枝较多、可增加地表郁闭度的生态应用型沙柳品系,奠定生物学研究基础。
The genes of Carotenoid Cleavage Dioxygenases-7(CCD7)and Carotenoid Cleavage Dioxygenases-8(CCD8)have important effects on the growth of lateral branches and roots of plants.In this study,the cDNA sequences of CCD7 and CCD8 were cloned from from S.psammophila,and the bioinformatics analysis,tissue expression specificity and prediction analysis were performed,which laid a foundation for the functional research and utilization of the CCD7 and CCD8 genes of S.psammophila,enriched the study of CCD gene family in woody plants.Salix purpurea,Populus trichocarpa Torr.&Gray homologous sequences were obtained from database phytozome 11 according to Arabidopsis,cucumber CCD7,CCD8 gene ID.ExPASy database was used to predict the physical,chemical characteristics and amino acid composition of protein.cNLS mapper was used to predict the nuclear localization signal(NLS)of protein.MEGA 6.0 and T-COFFEE were used to compare the homology of SpsCCD7 and SpsCCD8 protein sequences and construct phylogenetic trees.The cloned CDS were 1843 and 1674 bp,respectively,and were composed of 614 and 557 amino acid residues.SpsCCD7 and SpsCCD8 proteins had no transmembrane structure and no signal peptide region.Their theoretical isoelectric point(pI)and molecular weight were 7.55 and 6.10,respectively,69.2995 and 61.9815 kD were identified as hydrophilic proteins.Phylogenetic tree and amino acid sequence analysis showed that SpsCCD7 and SpsCCD8 were the most closely related to Populus trichocarpa Torr.&Gray and Salix purpurea,SpsCCD7 and SpsCCD8 have three typical conserved regions of RPE65 gene family,which belong to CCD protein family,but there are some differences at the end of the sequence.The SpsCCD7,SpsCCD8 gene of S.psammophila was expressed in root,flower,leaf,bud,shoot tip and tender stem of all tissues,and the highest expression level was found in shoot tip of S.psammophila quantitative analysis.The tissue-specific expression of S.psammophila is different from that of other woody plants,which may be related to S.psammophila itself being a shrub.This study successfully cloned CCD7 and CCD8 gene from S.psammophila which belong to the CCD gene family and which laid the foundation for further exploring the mechanism of woody plant CCD gene family regulation on branching,for the future cultivation of more bushy branches and which can increase the degree of surface canopy closure the ecologically applied S.psammophila strains laid the foundation for biological research.
作者
杨海峰
金娜
于兴旺
贺玉娇
李安玉
张鑫
刘振明
Yang Haifeng;Jin Na;Yu Xingwang;He Yujiao;Li Anyu;Zhang Xin;Liu Zhenming(College of Forestry,Inner Mongolia Agricultural University,Huhhot,010018)
出处
《分子植物育种》
CAS
北大核心
2021年第12期3948-3958,共11页
Molecular Plant Breeding
基金
国家科技重大专项项目(2018ZX08020002-005-0050)
国家自然基金项目(31660216)共同资助。