花生DOFH10在细胞中可能通过小泡运输并在网格蛋白相关途径被专一地识别（英文）

Cell Transport of DOFH10 in Arachis hypogaea L. Is Possibly via Vesicle and Specifically Recognized in Clathrin Related Pathway

为对花生未知的DOFH10基因进行研究,根据DOF转录调控因子DOF (DNA binding with One Finger)GW391728的c DNA序列,采用引物对s162-a1469和引物s30-a1469用PCR方法在花生16个品种中进行克隆比较,并进一步对DOFH10在数据库进行序列比对,对其一级结构酶切位点、三维空间结构、蛋白质表达等进行研究。结果表明,用引物对s162-a1469在5个品种中克隆得到2类DOFH10基因A和B类。它们分别与野生花生染色体A03上的XM_016100960. 2和野生花生染色体B03上的XM_016334585. 2序列完全相同。用引物s30-a1469从所有16个品种中得到相同的B类DOFH10基因,包括6个多粒果实品种。A类基因在4个多粒果实品种和1个2粒果实品种中存在。序列比对表明,A和B类DOFH10基因是不同基因编码的,它们分别位于家培花生的A和B基因组。B类DOFH10在DOF保守序列区域与野生花生染色体B01上编码网格蛋白集装相关蛋白XP_016199412. 2的一段基因剪辑后的RNA序列完全相同,DOFH10蛋白上多个肉豆蔻酰十四酰修饰位点的分布,这2个结果意味着该蛋白在细胞内的运输可能通过膜系统进行。蛋白质印迹分析表明,DOFH10在发育中子叶专一表达。花生DOFH10基因有A和B等2类,其中B类DOFH10存在于所有家培花生品种中,是必需基因,其蛋白可能通过Clathrin依赖的小泡运输途径运入细胞核。A类DOFH10更多存在于多粒家培花生品种中,可能与果实发育早期细胞分裂有关。

Study unknown DOFH10(DNA binding with one finger)genes in peanuts,cloning of DOFH10 genes with PCR methods according to cDNA of DOF transcription factor GW391728 with primers s162-a1469 and primers s30-a1469 from 16 different peanut cultivars of Arachis hypogaea L.,sequences comparison,analysis on enzyme site and 3D structure and Western Blot on DOFH10 proteins were conducted.Two types of DOFH10 genes were separated from genome DNA of 5 cultivars with primers s162-a1469,type A DOFH10 was only existed in four cultivars with multiple seeds per fruit and one cultivar with two seeds per fruit.They were identical to XM_016100960.2 from Arachis duranensis chromosome A03 and to XM_016334585.2 from Arachis ipaensis chromosome B03 respectively.With primers s30-a1469,only type B DOFH10 was cloned from all the 16 cultivars used including all the six cultivars with multiple seeds per fruit.Type B DOFH10 had identical RNA and protein sequences at DOF zinc finger region to that of putitive clathrin assembly protein XP_016199412.2 encoded by gene on chromosome B01 of Arachis ipaensis.Multiple possible myristoylation sites scattered on DOFH10 protein,both results suggested that transportation of DOFH10 was probable via vesicle membrane traffic related to clathrin.DOFH10 protein was specifically expressed in developing cotyledons displayed by Western Blot.A and B two types of DOFH10 genes existed in cultivated peanut.The type B was present in all 16 peanut cultivars,and was an essential gene,was possibly transported into nucleus through clathrin dependant vesicle pathway.The type A DOFH10 tended more appeared in cultivars with multiple seeds per fruit,probably related to cell division in early fruit development.