山药DoIPTs基因克隆及珠芽发芽期表达分析  

Cloning of DoIPTs Genes in Yam and Its Expression Analysis During Bulbil Germination

作　　者：唐文芳 徐升胜[1] 段延碧[2] 龙雯虹[1] 杨荣萍[1] 张雪梅[3] 孟金贵[1] TANG Wenfang;XU Shengsheng;DUAN Yanbi;LONG Wenhong;YANG Rongping;ZHANG Xuemei;MENG Jingui(College of Horticulture and Landscape,Yunnan Agricultural University,Kunming 650201,China;Agricultural Teaching Experiment Center,Yunnan Agricultural University,Kunming 650201,China;College of Agronomy and Biotechnology,Yunnan Agricultural University,Kunming 650201,China)

机构地区：[1]云南农业大学园林园艺学院,云南昆明650201 [2]云南农业大学农科教学实验中心,云南昆明650201 [3]云南农业大学农学与生物技术学院,云南昆明650201

出　　处：《华北农学报》2022年第1期27-34,共8页Acta Agriculturae Boreali-Sinica

基　　金：国家自然科学基金项目(31460322)。

摘　　要：为了克隆山药异戊烯基转移酶基因,预测该基因编码蛋白的结构和特性,并检测该基因在山药珠芽发芽期的表达特征,采用同源克隆的方法克隆山药IPT基因,使用生物信息学的方法预测蛋白质的序列和结构,通过实时荧光定量PCR检测基因的表达量。结果表明,在山药中克隆到了长为1398,1044,1349 bp的3条核酸序列,依次命名为DoIPT1、DoIPT5a和DoIPT5b,基因登录号依次是:MW353160、MW353016、MW353017,各基因的阅读框长度依次为1137,861,1011 bp,编码氨基酸长度分别为378,286,336 aa。分析氨基酸生物信息得知,DoIPTs属于不稳定外在膜亲水性蛋白,DoIPT5b存在2个信号肽,其余无信号肽;3个蛋白的二级结构预测与三级结构模型图相符;DoIPTs的编码区与其他植物的异戊烯基转移酶蛋白高度同源。DoIPTs编码区域显示,DoIPTs N端含有P-loop NTPase结构域GXXGXGKS(T),具有催化形成细胞分裂素的能力。实时荧光定量PCR分析结果表明,在22℃无外界水条件下珠芽发芽时表皮中DoIPT5a的表达量最高,在潮湿环境下DoIPT1和DoIPT5b基因的表达量最高;多效唑处理能够使DoIPTs基因的表达上调。结果为进一步研究山药DoIPTs基因的功能奠定了基础。The aim of this research was to clone IPT(Isopentenyl transferase,IPT)genes in yam,predict the structure and property of DoIPT proteins and identify the expression of DoIPTs during bulbil sprouting.DoIPTs were cloned by homologous cloning techniques.The structure and property of DoIPT proteins were predicted by bioinformatics analysis.Real-time PCR technique was applied to identify the expression of these genes.Three nucleic acid sequences of 1398,1044,1349 bp were obtained from yam.The three sequences were named DoIPT1,DoIPT5a and DoIPT5b,and the registration numbers of these genes were MW353160,MW353016 and MW353017,respectively.The reading frame lengths were 1137,861,1011 bp respectively,and the encoded amino acid lengths were 378,286,336 aa,respectively.Bioinformatics analysis showed that DoIPTs were unstable,hydrophilic and external family proteins.There were two signal peptides in DoIPT5b,and no signal peptide was found in the other two proteins.The secondary structures in the three proteins were consistent with the prediction models of the tertiary structures of three genes.The encoded regions of DoIPTs had high similarity with the isopentenyl transferase protein of other plants.Coding areas of DoIPTs showed that the N-terminal regions of DoIPTs contained the P-loop NTPase structural domain GXXGXGKS(T),which was able to catalyse cytokinin synthesis.The results of Real-time PCR technique showed that the expression of DoIPT5a in sprouting bulbils was highest under the condition of water shortage at 22℃;and the expression of DoIPT1 and DoIPT5b genes was the highest in a humid environment;the expression of DOIPTs genes raised with paclobutrazol treatment.These results laid a foundation for further study on the function of DoIPTs in yam.

关 键 词：山药 基因克隆 DoIPT基因 珠芽 多效唑 相对定量表达 

分 类 号：Q78[生物学—分子生物学] S632.03[农业科学—蔬菜学]