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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References

[1] Abdull Razis A F, Iori R, Ioannides C. The natural chemopreventive phytochemical R-sulforaphane is a far more potent inducer of the carcinogen-detoxifying enzyme systems in rat liver and lung than the S-isomer. International Journal of Cancer Journal International du Cancer, 2011, 128(12): 2775-2782.

[2] Fahey J W, Haristoy X, Dolan P M, Kensler T W, Scholtus I, Stephenson K K, Talalay P, Lozniewski A. Sulforaphane inhibits extracellular, intracellular, and antibiotic- resistant strains of Helicobacter pylori and prevents benzo[a]pyrene- induced stomach tumors. Proceedings of the National Academy of Sciences of the United States of America, 2002, 99(11): 7610-7615.

[3] Berger M S, Cooley M E, Abrahm J L. A pain syndrome-associated with large adrenal metastases in patients with lung-cancer. Journal of Pain and Symptom Management, 1995, 10(2): 161-166.

[4] Abbaoui B, Riedl K M, Ralston R A, Thomas-Ahner J M, Schwartz S J, Clinton S K, Mortazavi A. Inhibition of bladder cancer by broccoli isothiocyanates sulforaphane and erucin: Characterization, metabolism, and interconversion. Molecular Nutrition & Food Research, 2012, 56(11): 1675-1687.

[5] Dang Y M, Huang G, Chen Y R, Dang Z F, Chen C, Liu F L, Guo Y F, Xie X D. Sulforaphane inhibits the proliferation of the BIU87 bladder cancer cell line via IGFBP-3 elevation. Asian PacificJournal of Cancer Prevention: APJCP, 2014, 15(4): 1517-1520.

[6] Agyeman A S, Chaerkady R, Shaw P G, Davidson N E, Visvanathan K, Pandey A, Kensler T W. Transcriptomic and proteomic profiling of KEAP1 disrupted and sulforaphane-treated human breast epithelial cells reveals common expression profiles. Breast Cancer Research and Treatment, 2012, 132(1): 175-187.

[7] Chung F L, Conaway C C, Rao C V, Reddy B S. Chemoprevention of colonic aberrant crypt foci in Fischer rats by sulforaphane and phenethyl isothiocyanate. Carcinogenesis, 2000, 21(12): 2287-2291.

[8] Jiang X, Bai Y, Zhang Z, Xin Y, Cai L. Protection by sulforaphane from type 1 diabetes-induced testicular apoptosis is associated with the up-regulation of Nrf2 expression and function. Toxicology and Applied Pharmacology, 2014, 279(2): 198-210.

[9] Miao X, Bai Y, Sun W, Cui W, Xin Y, Wang Y, Tan Y, Miao L, Fu Y, Su G. Sulforaphane prevention of diabetes- induced aortic damage was associated with the up-regulation of Nrf2 and its down-stream antioxidants. Nutrition & Metabolism, 2012, 9(1): 84.

[10] Li Z, Galli U, Becker L E, Bruns H, Nickkolgh A, Hoffmann K, Karck M, Schemmer P. Sulforaphane protects hearts from early injury after experimental transplantation. Annals of transplantation: quarterly of the Polish Transplantation Society, 2013, 18: 558-566.

[11] Piao C S, Gao S, Lee G H, Kim do S, Park B H, Chae S W, Chae H J, Kim S H. Sulforaphane protects ischemic injury of hearts through antioxidant pathway and mitochondrial K(ATP) channels. Pharmacological Research: the Official Journal of the Italian Pharmacological Society, 2010, 61(4): 342-348.

[12] Brown A F, Yousef G G, Jeffrey E H, Klein B P, Wallig M A, Kushad M M, Juvik J A. Glucosinolate profiles in broccoli: Variation in levels and implications in breeding for cancer chemoprotection. Journal of the American Society for Horticultural Science, 2002, 127(5): 807-813.

[13] Farnham M W, Wilson P E, Stephenson K K, Fahey J W. Genetic and environmental effects on glucosinolate content and chemoprotective potency of broccoli. Plant Breeding, 2004, 123(1): 60-65.

[14] Fahey J W, Holtzclaw W D, Wehage S L, Wade K L, Stephenson K K, Talalay P. Sulforaphane bioavailability from glucoraphanin-rich broccoli: Control by active endogenous myrosinase. PLoS ONE, 2015, 10(11): e0140963.

[15] Liang H, Yuan Q P, Dong H R, Liu Y M. Determination of sulforaphane in broccoli and cabbage by high-performance liquid chromatography. Journal of Food Composition and Analysis, 2006, 19(5): 473-476.

[16] Li Z S, Liu Y M, Fang Z Y, Yang L M, Zhuang M, Zhang Y Y, Zhao W, Sun P T. Variation of sulforaphane levels in Broccoli (Brassica oleracea var. Italica) during flower development and the role of gene Aop2. Journal of Liquid Chromatography & Related Technologies, 2014, 37(9): 1199-1211.

[17] 李占省, 刘玉梅, 方智远, 杨丽梅, 庄木, 张扬勇, 吕红豪, 孙培田. 甘蓝和青花菜不同器官中莱菔硫烷含量差异研究. 核农学报, 2017, 31(3): 447-454.

LI Z S, LIU Y M, FANG Z Y, YANG L M, ZHUANG M, ZHANG Y Y, LÜ H H, SUN P T. Study on difference of sulforaphane in cabbage head and different organs of broccoli. Journal of Nuclear Agricultural Sciences, 2017, 31(3): 447-454. (in Chinese)

[18] 姚雪琴, 谢祝捷, 李光庆, 邱海荣, 李媛. 青花菜不同器官中4-甲基亚磺酰丁基硫苷及萝卜硫素含量分析. 中国农业科学, 2011, 44(4): 851-858.

YAO X Q,XIE Z J,LI G Q,QIU H R,LI Y. Analysis of glucoraphanin and sulforaphane contents in different organs of broccoli. China Agriculture Science, 2011, 44(4): 851-858 . (in Chinese)

[19] Sonderby I E, Geu-Flores F, Halkier B A. Biosynthesis of glucosinolates-gene discovery and beyond. Trends in Plant Science, 2010, 15(5): 283-290.

[20] Falk K L, Vogel C, Textor S, Bartram S, Hick A, Pickett JA, Gershenzon J. Glucosinolate biosynthesis: demonstration and characterization of the condensing enzyme of the chain elongation cycle in eruca sativa. Phytochemistry, 2004, 65(8): 1073-1084.

[21] Grubb C D, Abel S. Glucosinolate metabolism and its control. Trends in Plant Science, 2006, 11(2): 89-100.

[22] Wu S H, Lei J J, Chen G J, Chen H C, Cao B H, Chen C M. De novo Transcriptome assembly of Chinese kale and global expression analysis of genes involved in glucosinolate metabolism in multiple tissue. Frontiers in Plant Science, 2017, 8(27): 1-16.

[23] Chen S X, Glawischnig E, Jorgensen K, Naur P, Jorgensen B, Olsen C E, Hansen C H, Rasmussen H, Pickett J A, Halkier B A. CYP79F1 and CYP79F2 have distinct functions in the biosynthesis of aliphatic glucosinolates in Arabidopsis. Plant Journal, 2003, 33(5): 923-937.

[24] Reintanz B, Lehnen M, Reichelt M, Gershenzon J, Kowalczyk M, Sandberg G, Godde M, Uhl R, Palme K. Bus, a bushy arabidopsis CYP79F1 knockout mutant with abolished synthesis of short-chain aliphatic glucosinolates. Plant Cell, 2001, 13(2): 351-367.

[25] Hansen C H, Wittstock U, Olsen C E, Hick A J, Pickett J A, Halkier B A. Cytochrome P450CYP79F1 from Arabidopsis catalyzes the conversion of dihomomethionine and trihomomethionine to the corresponding aldoximes in the biosynthesis of aliphatic glucosinolates. Journal of Biological Chemistry, 2001, 276(14): 11078-11085.

[26] 王红波, 李成, 曹阳理惠, 李晶. CYP79F1基因对西兰花的遗传转化的研究. 植物研究, 2014, 34(4): 516-523.

WANG H B, LI C, CAO Y L H, LI J. Genetic Transformation of CYP79F1 Gene in Brassica oleracea var. italica. Plant Research, 2014, 34(4): 516-523. (in Chinese)

[27] Brown P D, Tokuhisa J G, Reichelt M, Gershenzon J. Variation of glucosinolate accumulation among different organs and developmental stages of Arabidopsis thaliana. Phytochemistry, 2003, 62(3): 471-481.

[28] Sharma M, Mukhopadhyay A, Gupta V, Pental D, Pradhan A K. Bju B. CYP79F1 Regulates synthesis of propyl fraction of aliphatic glucosinolates in oilseed mustard Brassica juncea: Functional validation through genetic and transgenic approaches. PLoS ONE, 2016, 11(2): e0150060.

[29] 李占省, 刘玉梅, 方智远, 杨丽梅, 庄木, 张扬勇, 袁素霞, 赵文, 刘二艳, 孙培田. 青花菜DH群体花球中莱菔硫烷含量的遗传效应分析. 园艺学报, 2012, 39(1): 101-108.

Li Z S, Liu Y M, Fang Z Y, Yang L M, Zhuang M, Zhang Y Y, Yuan S X, Zhao W, Liu E Y, Sun P T. Determination of sulforaphane by high performance liquid chromatography and genetic analysis of DH population in broccoli florets. Acta Horticulturae Sinica, 2012, 39(1): 101-108. (in Chinese)

[30] Tantikanjana T, Mikkelsen M D, Hussain M, Halkier B A, Sundaresan V. Functional analysis of the tandem-duplicated P450 genes SPS/BUS/CYP79F1 and CYP79F2 in glucosinolate biosynthesis and plant development by Ds transposition-generated double mutants. Plant Physiology, 2004, 135(2): 840-848.

[31] Li Z S, Liu Y M, Fang Z Y, Yang L M, Zhuang M, Zhang Y Y, LV H H. Development and identification of anti-cancer component of sulforaphane in developmental stages of broccoli (Brassic leracea var. italic L.). Journal of Food and Nutrition Research, 2016, 4(8): 490-497.

[32] Guo L P, Yang R Q, Gu Z X. Cloning of genes related to aliphatic glucosinolate metabolism and the mechanism of sulforaphane accumulation in broccoli sprouts under jasmonic acid treatment. Journal of the science of food and agriculture, 2016, 96(13): 4329-4336.

[33] Yan X F, Chen S X. Regulation of plant glucosinolate metabolism. Planta, 2007, 226(6): 1343-1352.

[34] Kopriva S, Gigolashvili T. Glucosinolate synthesis in the context of plant metabolism. Advances in Botanical Research, 2016, 80: 99-124.

[35] Luck K, Jia Q D, Huber M, Handrick V, Wong G K S, Nelson D R, Chen F, Gershenzon J, Kollner T G. CYP79 P450 monooxygenases in gymnosperms: CYP79A118 is associated with the formation of taxiphyllin in Taxus baccata. Plant Molecular Biology, 2017, 95(1-2): 169-180.

[36] Chaban C, Waller F, Furuya M, Nick P. Auxin responsiveness of a novel cytochrome p450 in rice coleoptiles. Plant Physiology, 2003, 133(4): 2000-2009.

[37] Bak S, Feyereisen R. The involvement of two P450 enzymes, CYP83B1 and CYP83A1, in auxin homeostasis and glucosinolate biosynthesis. Plant Physiology, 2001, 127(1): 108-118.

[38] Baik H Y, Juvik J, Jeffery E H, Wallig M A, Kushad M, Klein B P. Relating glucosinolate content and flavor of broccoli cultivars. Journal of Food Science, 2003, 68(3): 1043-1050.

[39] Rohr F, Ulrichs C, Mewis I. Variability of aliphatic glucosinolates in Arabidopsis thaliana (L.) - Impact on glucosinolate profile and insect resistance. Journal of Applied Botany and Food Quality-Angewandte Botanik, 2009, 82(2): 131-135.

[40] Rohr F, Ulrichs C, Schreiner M, Nguyen C N, Mewis I. Effects of two folivorous insect species on the aliphatic glucosinolate profile in Arabidopsis thaliana ecotype crosses. Mitt Deut Ges Allgem, 2012, 18: 219.

[41] Wittstock U, Kliebenstein D J, Lambrix V, Reichelt M, Gershenzon J. Glucosinolate hydrolysis and its impact on generalist and specialist insect herbivores. Recent Advances in Phytochemistry, 2003, 37: 101-125.

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