桂花15-顺式-ζ-胡萝卜素异构酶基因(Z-ISO)的克隆及表达分析

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摘要 15-顺式-ζ-胡萝卜素异构酶(15-cis-ζ-carotene isomerase, Z-ISO)可以催化9,15,9'-三顺式-ζ-胡萝卜素异构化生成9,9'-二顺式-ζ-胡萝卜素，Z-ISO基因是植物类胡萝卜素代谢途径中最后一个被鉴定的基因。为克隆桂花(Osmanthus fragrans) Z-ISO基因的ORF序列并研究其表达特征，本研究利用已构建的桂花花瓣转录组数据库中Unigene序列信息，结合PCR技术克隆得到桂花OfZ-ISO1(GenBank登录号: KX120175)和OfZ-ISO2(GenBank登录号: KX120176)基因ORF序列。生物信息学分析表明，OfZ-ISO1和OfZ-ISO2基因均含有长为1 107 bp的ORF，编码368个氨基酸残基。OfZ-ISO1和OfZ-ISO2蛋白均含有6个跨膜螺旋区和3个保守的异构酶功能作用所必需的活性位点(H152、H268和D296)。OfZ-ISO1和OfZ-ISO2与玉米(Zea mays)和拟南芥(Arabidopsis thaliana) Z-ISO1氨基酸序列有较高的相似性，系统进化树分析发现，OfZ-ISO1和OfZ-ISO2与芝麻(Sesamum indicum)Z-ISO亲缘关系最近，聚为同一小支。qRT-PCR结果显示，堰虹桂花序发育过程中，OfZ-ISO1基因在花蕾期花序中表达量较低，铃梗期其表达量急剧增加，初开期表达量保持不变，而盛开期表达量显著下降；OfZ-ISO2基因在花蕾期和铃梗期花序中表达量较低，随后表达量逐渐增加，于盛开期表达量达到最高。在桂花不同花色品种中，丹桂品种堰虹桂花色最深、花瓣类胡萝卜素总量最高，金桂品种金球桂次之，银桂品种玉玲珑花色最浅、花瓣类胡萝卜素总量最低；总体上铃梗期和初开期时玉玲珑OfZ-ISO1和OfZ-ISO2基因的表达量较高，盛开期3个不同花色品种花瓣中OfZ-ISO1和OfZ-ISO2基因的表达量大致相同。本研究结果表明，桂花花序开放过程中OfZ-ISO1和OfZ-ISO2基因的表达水平对其类胡萝卜素的积累十分重要，不同花色品种类胡萝卜素含量与其OfZ-ISO1和OfZ-ISO2基因表达量并非正相关。研究结果将为全面揭示桂花呈色机制提供理论基础。

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关键词 ：桂花, 花色, 类胡萝卜素, 15-顺式-ζ-胡萝卜素异构酶基因(Z-ISO)

Abstract：15-cis-ζ-carotene isomerase (Z-ISO) can isomerize 9,15,9'-tri-cis-ζ-carotene into 9,9'-di-cis-ζ-carotene, and Z-ISO gene is the last identified gene in plant carotenoid biosynthetic pathway. In order to clone the ORF of Z-ISO genes in Osmanthus fragrans and study their expression profile, ORF sequences of OfZ-ISO1 (GenBank accession: KX120175) and OfZ-ISO2 (GenBank accession: KX120176) from O. fragrans were amplified using PCR based on the sequence information of Unigene from the petal transcriptome database of O. fragrans Yanhong Gui from the Aurantiacus Group. Bioinformatics analysis indicated that both OfZ-ISO1 and OfZ-ISO2 contained 1 107 bp ORF, encoding 368 amino acid residues. Six predicted transmembrane domains were in both OfZ-ISO1 and OfZ-ISO2 proteins, as well as 3 conserved residues (H152, H268 and D296) which were essential for isomerase activity. OfZ-ISO1 and OfZ-ISO2 from O. fragrans shared high sequence similarity with ZmZ-ISO1 from Zea mays and AtZ-ISO1 from Arabidopsis thaliana. Phylogenetic analysis showed that OfZ-ISO1 and OfZ-ISO2 were gathered into one branch with Sesamum indicum Z-ISO, which revealed that OfZ-ISO1 and OfZ-ISO2 showed the closest genetic relationship with Z-ISO from S. indicum. Gene expression showed that during the flower development of O. fragrans Yanhong Gui, the expression level of OfZ-ISO1 was low in the inflorescences at bud stages, dramatically increased at linggeng stage, kept the same at initial flowering stage and greatly decreased at full flowering stage, while that of OfZ-ISO2 was low in the inflorescences at bud stages and linggeng stage, then gradually increased, and reached the maximum at full flowering stage. In the O. fragrans cultivars with different petal color, Yanhong Gui from the Aurantiacus Group showed the darkest petal color with the lowest L* value and the highest a* value and the highest total carotenoid content. In Jin Qiugui from the Luteus Group, the petal color was lighter than that of Yanhong Gui from the Aurantiacus Group and the total carotenoid content was lower than that of Yanhong Gui, and Yu Linglong from the Albus Group showed the lightest petal color with the highest L* value and the lowest a* value and the lowest total carotenoid content. Surprisingly, the expression levels of OfZ-ISO1 and OfZ-ISO2 were not the highest in Yanhong Gui with the highest total carotenoid content. Generally, the expression levels of OfZ-ISO and OfZ-ISO were the highest in Yu Linglong at linggeng stage and initial flowering stage, however, at full flowering stage, the expression levels were the same in the 3 cultivars with different petal color. The results of this study revealed that the expression levels of OfZ-ISO1 and OfZ-ISO2 during flower opening were essential to the carotenoid accumulation of O. fragrans, and total carotenoid content of cultivars with different petal color was not positively related with the expression level of OfZ-ISO1 and OfZ-ISO2. This study has important practical significance to fully reveal the mechanism of petal coloration in O. fragrans.

Key words： Osmanthus fragrans Petal color Carotenoids 15-cis-ζ-carotene isomerase gene (Z-ISO)

收稿日期: 2016-03-31 出版日期: 2016-08-06

基金资助:国家自然科学基金;浙江省自然科学基金

通讯作者: 赵宏波 E-mail: zhaohb@zafu.edu.cn

引用本文:

张超罗云王艺光付建新赵宏波. 桂花15-顺式-ζ-胡萝卜素异构酶基因(Z-ISO)的克隆及表达分析[J]. , 2016, 24(10): 1512-1521.

链接本文:

http://journal05.magtech.org.cn/Jwk_ny/CN/ 或 http://journal05.magtech.org.cn/Jwk_ny/CN/Y2016/V24/I10/1512

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