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

Abstract

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

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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Full Length Cloning, Expression and Correlation Analysis of P450 CYP79F1 Gene with Sulforaphane Content in Different Broccoli Organs

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