芒果类胡萝卜素裂解双加氧酶1(CCD1)基因的克隆与序列分析

Cloning and Sequence Analysis of Carotenoid Cleavage of Dioxygenase 1 (CCD1) Gene from Mango(Mangifera indica)

类胡萝卜素裂解双加氧酶(carotenoid cleavage dioxygenases,CCD)是植物类胡萝卜素新陈代谢中的降解酶,类胡萝卜素在双加氧酶的作用下氧化产生具有生物活性的不同衍生物,例如维生素A,植物激素脱落酸以及芳香物质,这些物质在植物的颜色、苦味、香气方面具有重要意义。通过转录组数据设计CCD1基因的全长引物,采用RT-PCR技术从芒果(Mangifera indica)果皮中克隆到类胡萝卜素裂解双加氧酶CCD1的全长序列。结果表明:c DNA序列长度为1 900 bp,开放阅读框为1 614 bp,共编码537个氨基酸。CCD1蛋白属于稳定蛋白,在CCD1编码的蛋白质的氨基酸各组分中,亮氨酸(Leu)含量为最高,缬氨酸(Val)的含量次之。蛋白序列分析表明芒果中的CCD1基因编码的蛋白存在一个结构域RPE65,并且属于RPE65 superfamily家族。通过构建系统发生树,结果发现芒果CCD1基因所编码的蛋白序列与蜜柑、甜橙、栗子、大豆等的蛋白序列亲缘关系较近,可聚为一类,而与咖啡豆、菠菜、马铃薯、烟草的亲缘关系较远。芒果的CCD1与柑橘的CCD1的一致性为90%,有比较近的进化关系。亚细胞定位分析结果显示,芒果CCD1定位在细胞质中。对芒果的CCD1蛋白进行疏水性/亲水性预测,结果发现氨基酸疏水/亲水性主要介于+2.267^-0.633之间,CCD1蛋白属于亲水性蛋白。分析二级结构和三级结构得出CCD1基因编码的蛋白质主要由α-螺旋和无规则卷曲构成了其二级结构,α-螺旋占22.16%,无规则卷曲占37.43%。

Carotenoid cleavage dioxygenases （CCD） is the adegradation enzyme in the metabolism of carotenoids in plant. Carotenoids produce different derivatives with biological activities under the treatment of dioxygenases, such as vitamin A, plant hormone abscisic acid and aromatic substances. These substances play vital roles in color, taste, aroma of plants The full-length primers of CCD1 gene were designed by transcriptome data and RT-PCR wasused to clone the full-length sequence of CCDI gene from Mangifera indica. The results showed that the length of cDNA sequence was 1 900 bp and the ORF was 1 614 bp, encoding 537 amino acids. CCD1 protein was a stable protein. In the amino acids components of the protein encoded by CCDI, the content of Leu was the highest, followed by the content of Val. The protein sequence analysis indicated that the protein encoded by CCDI gene in mango had a structural domain called RPE65, which belonged to RPE65 superfamily. By constructing the phylogenetic tree, we found that the protein sequence encoded by mango CCDI gene was close to that of honey, orange, chestnut, soybeans, and could be clustered into one category. However, this genetic rclationship was far away with coffee beans, spinach, potato and tobacco. Since the consistency of CCDI from mango and citrus was 90%, there was a relatively close evolutionary relationship. The results of subcellular localization analysis demon- strated that the CCDI of mango was located in the cytoplasm. The hydrophobicity and hydrophilicity of CCDI protein of mango were predicted, the results of which suggested that the hydrophobicity/hydrophilicity of amino acids was mainly between ＋2.267-0.633 and CCDI protein belonged to hydrophilic protein. The analysis of secondary structure and tertiary structure found that the protein encoded by CCDI gene mainly consisted of α-helix and irregular curl, and the α-helix and irregular curl accounted for 22.16% and 37.43%, respectively.