青花菜P450CYP79F1全长克隆、表达及其与不同器官中莱菔硫烷含量的相关性分析

doi:10.3864/j.issn.0578-1752.2018.12.012

摘要/Abstract

摘要： 【目的】克隆青花菜细胞色素家族P450CYP79F1，分析其序列特征，阐明其在不同发育时期器官中的表达情况及其与莱菔硫烷含量的关系，为进一步揭示莱菔硫烷合成机理提供科学依据，为青花菜品种改良和新品种选育奠定基础。【方法】通过5′和3ˊ端RACE克隆技术获得青花菜CYP79F1全长序列，利用DNAMAN 6.0进行基因拼接、氨基酸序列分析和蛋白二级结构预测，结合在线分析程序和软件进行基因和编码蛋白生物信息学分析；利用荧光定量PCR技术研究CYP79F1在不同发育器官中的表达情况；结合HPLC方法测定各相应时期器官中莱菔硫烷含量，包括青花菜发育时期的根、茎、叶，成熟花球，抽薹期花蕾（顶端蕾、成熟蕾、开花前1 d的蕾和花）和种子；对CYP79F1表达量与和莱菔硫烷含量进行相关性分析。【结果】获得了CYP79F1全长序列，GenBank登录号为MG012890，该基因全长2 014 bp，包含一个1 620 bp的开放读码框（ORF），编码540个氨基酸；编码的蛋白与甘蓝、白菜、芥蓝和油菜等同源蛋白的相似性均在95%以上；生物信息学分析表明该基因编码的酶蛋白有2个信号肽和2个跨膜结构域，为亲水性稳定蛋白，Wolf Psort预测其亚细胞定位于细胞质中；CYP79F1在根和茎中的表达量较高，叶中表达量最低，在花器官发育时期，自顶端蕾到花发育过程中呈现逐渐降低的表达趋势，种子中该基因表达量与花处于同一水平。Pearson相关性分析显示，青花菜发育时期的根、茎、叶、成熟花球、抽薹期花蕾和种子中莱菔硫烷含量与CYP79F1表达量无显著相关关系，但抽薹期花蕾自顶端花蕾到花中莱菔硫烷生成量与CYP79F1表达量呈显著正相关关系（R=0.96，P<0.05），CYP79F1在调控莱菔硫烷的生成方面起重要作用，尤其是在生殖器官花蕾的发育过程中，能够显著上调莱菔硫烷的生成量。【结论】CYP79F1在青花菜不同发育器官中对莱菔硫烷的生成发挥着重要调控作用，可能与青花菜不同器官中莱菔硫烷含量多样性密切相关。

关键词: 青花菜, 莱菔硫烷, CYP79F1, 表达分析, HPLC

Abstract: 【Objective】The aim of this study was to clone the full length of P450 family CYP79F1 gene from broccoli, investigate its sequence characteristic and analyze its expression and correlation with sulforaphane content in different developmental broccoli organs. These findings will provide a scientific basis for further revealing the mechanism of sulforaphane metabolism in broccoli and lay a practical foundation for the genetic improvement and breeding of broccoli varieties. 【Method】 The full length sequence of CYP79F1 gene was obtained by RACE cloning (5′ and 3′). According to the software of DNAMAN 6.0, gene splicing, amino acid sequence analysis and protein secondary structure prediction of CYP79F1 gene, at the same time, bioinformatics information was also predicted by online program and software. The quantitative real-time PCR (qRT-PCR) method was used to study the expression of CYP79F1 gene in different developmental organs of broccoli. The sulforaphane contents of different developmental organs were detected by high performance liquid chromatography (HPLC) method, and they were roots, stems, leaves, developmental buds at bolting stage (top buds, mature buds, buds one day before flowering and flowers), and seeds. Pearson correlation analysis was carried out by the software SPSS 10.0.【Result】Full length sequence of CYP79F1 gene were cloned, and the gene of CYP79F1 was 2 014 bp in length, containing a 1 620 bp opening reading frame (ORF), encoded a polypeptide of 540 amino acids. They shared over 95% identity with the homologous proteins from Brassica plants, such as cabbage, Chinese cabbage, Chinese kale and rape. The bioinformatics analysis showed that the CYP79F1 protein was hydrophilic protein with two signal-peptides and two transmembrane regions, and the Wolf Psort protection indicated that they were located in the cytoplasm. The expression of CYP79F1 gene was higher in the roots and the stems, and the lowest in the leaves. During developmental stage of buds, the expression of CYP79F1 gene was up-regulated in the early (young buds), and then decreased (mature buds to flowers). There was no difference significance in gene expression between roots, stems, leaves, developmental buds at bolting stage and seeds. Correlation analysis showed that the expression level of CYP79F1 gene showed an extremely significant positive correlation with sulforaphane content in developmental buds at bolting stage (R=0.96，P<0.05), and the expression of CYP79F1 gene might influence the generation of sulforahane in different organs of broccoli, especially in developmental buds.【Conclusion】The CYP79F1 gene were comprehensively obtained and characterized from broccoli, CYP79F1 may play an important role in sulforaphane metabolism and revealing the diversity of sulforaphane content in different organs of broccoli.

Key words: broccoli, sulforaphane, CYP79F1, expression analysis, HPLC

李占省，刘玉梅，方智远，杨丽梅，庄木，张扬勇，吕红豪. 青花菜P450CYP79F1全长克隆、表达及其与不同器官中莱菔硫烷含量的相关性分析[J]. 中国农业科学, 2018, 51(12): 2357-2367.

LI ZhanSheng, LIU YuMei, FANG ZhiYuan, YANG LiMei, ZHUANG Mu, ZHANG YangYong, Lü HongHao. Full Length Cloning, Expression and Correlation Analysis of P450 CYP79F1 Gene with Sulforaphane Content in Different Broccoli Organs[J]. Scientia Agricultura Sinica, 2018, 51(12): 2357-2367.

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