药西瓜UDP-糖基转移酶基因的克隆与表达分析

为明确药西瓜UDP-糖基转移酶催化葫芦素形成葫芦素配糖体的表达规律,以药西瓜品种"WM9"叶片为供试材料,采用RT-PCR技术克隆药西瓜UDP-糖基转移酶基因,并对编码蛋白进行分析。通过实时荧光定量PCR(qRT-PCR),以ACTIN为内参基因,分析获得的2个药西瓜UDP-糖基转移酶基因在不同组织器官中的表达规律。结果表明:克隆得到2个UDP-糖基转移酶基因,分别为1 546 bp和1 559 bp的cDNA全长序列,命名为UDP-E1和UDP-E2。对扩增获得的序列进行生物学信息分析,确定UDP-E1基因的完整开放阅读框(ORF)为1 314 bp,可编码氨基酸437个,理论分子量为49.02 ku,等电点为5.99,属稳定蛋白,Genbank登录号MK576125。UDP-E2基因的ORF为846 bp,可编码氨基酸281个,理论分子量为32.78 ku,等电点为5.23,属不稳定蛋白,Genbank登录号MK576126。这2个基因属于糖基转移酶超家族。UDP-E1和UDP-E2均与香瓜、黄瓜的UDP-糖基转移酶基因序列相似性最高。在各组织器官中,UDP-糖基转移酶均有表达,且在茎中表达水平最低,叶中表达水平最高。

Cloning and expression analysis of UDP-Glycosyl transferase gene from Citrullus colocynthis L.Shrad

To clarify the expression pattern of cucurbitacin glycoside catalyzed by Citrullus colocynthis L.Shrad UDP-glycosyl transferase.The C.colocynthis L.Shrad "WM9" variety leaves were used as the test materials.The C.colocynthis L.Shrad UDP-glycosyl transferase gene was cloned by RT-PCR and the physicochemical properties of the encoded proteins were analyzed.The expression patterns of two C.colocynthis L.Shrad UDP-glycosyl transferase genes in different tissues and organs were investigated by qRT-PCR and ACTIN as the internal reference gene.The results showed that:Two UDP-glycosyl transferase genes UDP-E1 and UDP-E2 with full-length cDNA sequences of 1 546 bp and 1 559 bp were obtained,named.The results of biological information analyses determined that the complete open reading frame (ORF) of the UDP-E1 gene is 1 314 bp in length encoding 437 amino acids,and its theoretical molecular weight is 49.02 ku.The isoelectric point was 5.99 and is a stable protein.Genbank accession number is MK576125.The UDP-E2 gene has an ORF of 846 bp and encodes 281 amino acids with a theoretical molecular weight of 32.78 ku and an isoelectric point of 5.23.It is an unstable protein and Genbank accession number is MK576126.These two genes belong to the glycosyl transferase superfamily.Sequence multiplex alignment and phylogenetic tree analysis showed that the genes UDP-E1 and UDP-E2 have the highest similarity to the UDP-glycosyl transferase gene sequences of melon and cucumber.UDP-glycosyl transferase was expressed in all tissues and organs,and the expression level was the lowest in stems and the highest in leaves.The expression level was the lowest in stems and the highest in leaves.