Boonkaew Tippawan, Mongkolsiriwatana Chareerat, Vongvanrungruang Ananya, Srikulnath Kornsorn, Peyachoknagul Surin
Department of Genetics, Faculty of Science, Kasetsart University, 50 Ngamwongwan, Chatuchak, Bangkok, 10900, Thailand.
Department of Genetics, Faculty of Liberal Arts and Science, Kasetsart University, Kamphaengsaen Campus, Nakhon Pathom, 73140, Thailand.
Genes Genomics. 2018 Jul;40(7):735-745. doi: 10.1007/s13258-018-0682-4. Epub 2018 Mar 23.
Coconuts (Cocos nucifera L.) are divided by the height into tall and dwarf types. In many plants the short phenotype was emerged by mutation of the GA20ox gene encoding the enzyme involved in gibberellin (GA) biosynthesis. Two CnGA20ox genes, CnGA20ox1 and CnGA20ox2, were cloned from tall and dwarf types coconut. The sequences, gene structures and expressions were compared. The structure of each gene comprised three exons and two introns. The CnGA20ox1 and CnGA20ox2 genes consisted of the coding region of 1110 and 1131 bp, encoding proteins of 369 and 376 amino acids, respectively. Their amino acid sequences are highly homologous to GA20ox1 and GA20ox2 genes of Elaeis guineensis, but only 57% homologous to each other. However, the characteristic amino acids two histidines and one aspartic acid which are the two iron (Fe) binding residues, and arginine and serine which are the substrate binding residues of the dioxygenase enzyme in the 20G-FeII_Oxy domain involved in GA biosynthesis, were found in the active site of both enzymes. The evolutionary relationship of their proteins revealed three clusters in vascular plants, with two subgroups in dicots and three subgroups in monocots. This result confirmed that CnGA20ox was present as multi-copy genes, and at least two groups CnGA20ox1 and CnGA20ox2 were found in coconut. The nucleotide sequences of CnGA20ox1 gene in both coconut types were identical but its expression was about three folds higher in the leaves of tall coconut than in those of dwarf type which was in good agreement with their height. In contrast, the nucleotide sequences of CnGA20ox2 gene in the two coconut types were different, but the expression of CnGA20ox2 gene could not be detected in either coconut type. The promoter region of CnGA20ox1 gene was cloned, and the core promoter sequences and various cis-elements were found. The CnGA20ox1 gene should be responsible for the height in coconut, which is different from other plants because no mutation was present in CnGA20ox1 gene of dwarf type coconut.
椰子（Cocos nucifera L.）根据高度分为高种和矮种类型。在许多植物中，短表型是由编码参与赤霉素（GA）生物合成的酶的GA20ox基因突变产生的。从高种和矮种椰子中克隆了两个CnGA20ox基因，即CnGA20ox1和CnGA20ox2，并对它们的序列、基因结构和表达进行了比较。每个基因的结构由三个外显子和两个内含子组成。CnGA20ox1和CnGA20ox2基因的编码区分别为1110和1131 bp，分别编码369和376个氨基酸的蛋白质。它们的氨基酸序列与油棕的GA20ox1和GA20ox2基因高度同源，但彼此之间只有57%的同源性。然而，在两种酶的活性位点都发现了特征性氨基酸，即参与GA生物合成的20G-FeII_Oxy结构域中作为两个铁（Fe）结合残基的两个组氨酸和一个天冬氨酸，以及双加氧酶的底物结合残基精氨酸和丝氨酸。它们蛋白质的进化关系在维管植物中显示出三个聚类，双子叶植物中有两个亚组，单子叶植物中有三个亚组。这一结果证实CnGA20ox以多拷贝基因形式存在，并且在椰子中至少发现了两组CnGA20ox1和CnGA20ox2。两种椰子类型中CnGA20ox1基因的核苷酸序列相同，但其在高种椰子叶片中的表达量比矮种椰子叶片中的表达量高约三倍，这与其高度情况相符。相反，两种椰子类型中CnGA20ox2基因的核苷酸序列不同，但在两种椰子类型中均未检测到CnGA20ox2基因的表达。克隆了CnGA20ox1基因的启动子区域，并发现了核心启动子序列和各种顺式作用元件。CnGA20ox1基因应该是决定椰子高度的原因，这与其他植物不同，因为矮种椰子的CnGA20ox1基因没有突变。
