蒜头果中CYP71基因克隆与果实不同发育时期的表达分析

Cloning of CYP71 gene in Malania oleifera and expression analysis in different developmental periods

关键词:  蒜头果, CYP71基因, 氰苷, 生物信息学分析, 基因表达

Key words:  Malania oleifera, CYP71 gene, cyanogenic glucosides, bioinformatics analysis, gene expression

摘要:

由氨基酸衍生的氰苷是由植物细胞色素P450单加氧酶(CYP)催化氨基酸生成的次生代谢产物,与植物的防御和抗逆境胁迫相关。该研究通过分析蒜头果转录组数据,从中分离并克隆得到1条细胞色素P450基因(命名为MoCYP71,GenBank登录号为MK172858),对其进行生物信息学分析并检测该基因在果实不同发育时期的表达情况。结果表明:MoCYP71基因含1 572 bp,编码523个氨基酸,该基因的cDNA序列与咖啡(Coffea eugenioides,XM_027319282)、小果咖啡(Coffea arabica,XM_027213456)中的CYP71基因的mRNA序列均有88%的一致性; MoCYP71蛋白的相对分子质量为58 976.54,理论等电点pI为8.10,分子式为C₂₆₇₅H₄₁₈₄N₇₀₄O₇₄₄S₂₇,不稳定系数(Ⅱ)为40.84,是一种不稳定蛋白; 该蛋白不存在信号肽,存在于分泌途径,含有两个跨膜结构,分别位于20～37和311～333位的氨基酸为跨膜疏水螺旋,锚定于细胞器上; 该蛋白含有CYP家族的保守结构域,包括脯氨酸富集区(PPSPPRLP)、K螺旋(KETFR)、I螺旋(GGIDTS)、PERF域(PERF)和识别结构域血红素结合域(FGAGRRICPG),与可可、榴莲、高粱等的CYP71E家族的蛋白(GenBank登录号分别为EOX92908.1、XP_022773875.1和AAC39318.1)聚为一支; 在花谢后,MoCYP71基因表达量逐渐降低, 花谢后1个月>2个月>3个月,但在花谢后4个月的表达量急剧增加。以上结果对研究蒜头果的对虫害的防御、组织成熟及蒜头果中有效次生代谢产物的发掘具有重要意义。

Abstract:

Amino acids-derived cyanogenic glucosides catalyzing by plant cytochrome P450 enzymes are plant secondary metabolism, which is related to plant defense and anti-stress. A gene of cytochrome P450(namely MoCYP71, GenBank ID is MK172858)from Malania oleifera was isolated and cloned through analyzing the transcriptome of M. oleifera, then its function was analyzed by bioinformatics analysis, and the expression of MoCYP71 in different developmental periods of fruit was detected. The results were as follows: MoCYP71 gene has 1 572 bp, encodes 523 amid acids, and its sequence of cDNA has 88% identity with the CYP71 cDNA of Coffea eugenioides(XM_027319282)and Coffea arabica(XM_027213456); The relative molecular weight of MoCYP71 protein was 58 976.54, the theoretical pI was 8.10, the molecular formula was C₂₆₇₅H₄₁₈₄N₇₀₄O₇₄₄S₂₇, the instability index(Ⅱ)was 40.84, and the protein belonged to an instable protein; There had no siginal peptide in this protein sequence, and the protein exists in the secretion pathway and contained two transmembrane structure, located in the protein sequences of 20-37 and 311-333, and the transmembrane sites anchored in the organelle membrane; MoCYP71 had all the conserved domain of CYP family, containing proline-rich region(PPSPPRLP), K helix region(KETFR), I helix region(GGIDTS), PERF domain(PERF)and the main identified feature heme-binding region(FGAGRRICPG), and the protein MoCYP71 and the CYP71E protein in Theobroma cacao, Durio zibethinus, Sorghum bicolor(GenBank ID is EOX92908.1, XP_022773875.1and AAC39318.1, respectively)clustered into one clan; The expression level of MoCYP71 gene reduced gradually in the first three months after blossom fading, particularly, 1st month the fruit after blossom fading> 2nd month> 3rd month, however, its expression level increased sharply in 4th month. The present study had a important significance to defense to insect pests, tissue ripening and secondary metabolism digging in Malania oleifera.