摘要
青花菜(Brassica oleracea var. italica)，又名西兰花、西班牙芥蓝等，是十字花科（Cruciferae）芸薹属（Brassica）一、二年生草本植物，甘蓝类蔬菜的一个变种。青花菜富含硫代葡萄糖苷（Glucosinolate，GS，简称“硫甙”）成分4-甲基亚磺酰基丁基硫甙（Glucoraphanin，RAA），其水解产物莱菔硫烷（Sulforaphane，SF）是迄今为止在蔬菜中发现的抗癌活性最强的活性成分之一。研究发现，莱菔硫烷能够显著降低肝癌、胃癌、肺癌、乳腺癌、膀胱癌等多种癌的患病率，同时能够预防心脑血管疾病的发生。目前，国内外对莱菔硫烷的医学价值报道较多，而对甘蓝类蔬菜作物中莱菔硫烷含量水平和资源筛选，青花菜整个生育过程中莱菔硫烷含量变化规律，青花菜花球中莱菔硫烷含量的遗传效应、QTL定位和相关基因在青花菜中的表达调控研究鲜有报道。
本研究以甘蓝类蔬菜变种（甘蓝、青花菜、芥蓝和苤蓝），青花菜DH群体（176份）和六世代群体（P1、P2、F1、B1、B2和F2）为材料，对甘蓝类蔬菜变种的可食性器官和非可食性部分中莱菔硫烷含量进行了检测和分析，研究了青花菜整个生长发育过程不同器官中莱菔硫烷含量变化规律，青花菜DH群体和六世代群体花球中莱菔硫烷含量的遗传效应，构建了基于青花菜DH永久群体的较高密度的遗传连锁图谱，并对青花菜花球中莱菔硫烷含量进行了QTL定位，最后对青花菜莱菔硫烷含量相关调控基因进行了RT-PCR表达分析和相关性分析，构建了CYP79F1和AOP2超表达和RNAi表达载体。
主要研究结果如下：
（1）构建了用于测定甘蓝类蔬菜中莱菔硫烷成分的RP-HPLC检测体系和UPLC-MS/MS鉴定体系，并采用正交设计L9（34）优化了莱菔硫烷提取方法。结果表明采用梯度洗脱法检测莱菔硫烷成分，在标准浓度范围内线性方程良好：Y=3.69e-004X-1.26（R2=0.9994），回收率为96.2%（n=6），精密度良好（RSD=2.50e-9%，n=6）。UPLC-MS/MS成分鉴定表明，样品中莱菔硫烷成分与标准样品在保留时间（2.17min）和分子量等方面一致，确保了HPLC定量的准确性。
（2）对甘蓝类蔬菜不同变种材料：48份甘蓝材料的叶球，29份青花菜自交系的花球、幼茎和叶片，44份芥蓝材料（自交系和F1）的花薹和39份苤蓝（自交系和F1）球茎进行了莱菔硫烷含量的HPLC分析。结果表明，各变种材料中莱菔硫烷含量最高的为青花菜花球，其余从高到低依次是芥蓝花薹，苤蓝球茎和甘蓝叶球；青花菜材料中莱菔硫烷含量最高的部分为花球，其次是幼茎，叶片最低，获得了14份高莱菔硫烷含量的变种材料。
（3）以4份青花菜材料，包括自交系B691和B692及其F1材料B693（B692×B691）和B694（B691×B692），研究了青花菜整个生长发育过程中莱菔硫烷含量变化规律。首次探明了青花菜在发育过程中莱菔硫烷含量在不同器官中呈现不同的变化规律。成熟种子、抽薹期花蕾、定植后20d时的叶片和早期芽苗中莱菔硫烷含量较高，具有较大的开发价值。
（4）以莱菔硫烷含量差异显著两个青花菜自交系86101和90196配制的F1，然后利用游离小孢子培养的方法构建了包含176份DH系的永久群体，并构建了六世代群体（P1、P2、F1、B1、B2和F2）。采用高效液相色谱法（HPLC）对青花菜群体花球中莱菔硫烷含量进行了测定，运用植物数量性状主基因+多基因混合遗传模型对两群体中莱菔硫烷含量进行了遗传效应分析。结果表明，青花菜DH群体花球中莱菔硫烷含量受3对主基因-加性-上位性+多基因控制（G-1），群体主基因遗传率为89.28%，多基因遗传率为2.58%，主基因遗传率较高，表明该性状主要受主基因调控。采用主基因+多基因混合分离分析遗传模型对六世代群体花球中莱菔硫烷含量进行分析，表明该性状符合E-1-0遗传模型：2对加性-显性-上位性主基因+加性-显性-上位性多基因模型，主基因和多基因遗传率几乎各占50%，表明该性状受主基因和多基因共同调控。
（5）以青花菜DH群体（176份DH系）为材料，从3786对SSR引物（EST-SSR和gSSR）中筛选出314对多态性好，且在双亲间有差异、重复性好、稳定性高的SSR引物，采用Joinmap4.0软件构建了一个覆盖基因组全长1293.32cM，包含268对SSR引物，分布于9条连锁群的遗传连锁图谱。该图谱分子标记间平均遗传距离为4.8cM，最长的连锁群为LG1，覆盖基因组长度为302.8cM，包含60对SSR引物，标记间平均遗传距离为5.1cM；最短的连锁群为LG7，覆盖基因组长度为26.9cM，包含16对SSR引物，标记间平均遗传距离为1.7cM，该连锁群的分子标记间平均遗传距离最短。
（6）根据青花菜DH群体花球中莱菔硫烷含量的测定结果和遗传连锁图谱的构建，采用统计学软件SPSS10.0和MapQTL4.0软件对青花菜DH群体花球中莱菔硫烷含量进行了QTL定位。结果显示，采用区间作图（Interval mapping，IM）和MQM（Multiple QTL Mapping）法作图，共检测到13个QTL（SF1~13），分布在4条连锁群上，分别为LG1、LG6、LG8和LG9，各连锁群上分布的QTL分别为3、2、1和7个。
（7）以青花菜B108为试验材料对AOP2基因进行了巢式PCR扩增，获得了一条长度为1696bp的序列。将该序列在NCBI上经Blastx分析，结果发现15条序列与该片段相似性在73%以上，其中相似性在95%以上的有14条，相似性为100%的有3条。采用MEGA对相似性较高的序列构建了MP和NJ发育树，表明AOP2在芸薹属作物中存在不同的拷贝数，多样性较为丰富。同时将该克隆片段在甘蓝和白菜基因组中进行了Blastx分析和染色体锚定，结果发现该序列在甘蓝和白菜基因组中分别有3个不同的拷贝，分布于甘蓝的第3、第8和第9染色体上，在白菜上分别位于第2、第3和第9染色体上，相似性都在90%以上。
（8）对青花菜B108不同发育时期的花器官，包括商品期的花球，抽薹期的顶端花蕾、成熟蕾、开花前1d花蕾和完全开放的花及成熟种子中莱菔硫烷含量进行了HPLC分析，同时与两个相关基因CYP79F1和AOP2在相应器官上的RT-PCR（反转录PCR）表达量进行相关性分析。结果表明，青花菜B108商品期的花球，抽薹期的顶端花蕾、成熟蕾、开花前1d花蕾和完全开放的花，成熟种子中莱菔硫烷含量差异显著（P<0.05）。RT-PCR表达结果显示，不同的花器官发育时期AOP2和CYP79F1基因的表达量差异极显著（P<0.01），AOP2和CYP79F1基因在花中的表达量均处于最高水平。各时期花器官中莱菔硫烷含量与两个相关基因的表达量相关性分析结果表明两基因之间的表达量呈正比关系，而与莱菔硫烷的生成量呈反比关系。
（9）构建了AOP2基因的RNAi表达载体和CYP79F135S强启动子超表达载体。表达载体pJG081-AOP2在植物中表现为Hyg抗性，在细菌中表现为Kan抗性；CYP79F1基因的两个超表达载体（010和291）均带有EGFP（绿色荧光表达蛋白）报告基因，这为验证两基因的功能奠定了基础。
Broccoli (Brassica oleracea var. italica) is rich in glucosinolates (GCs), and it is one variation ofBrassica which has one or two year’s life time. Glucoraphanin (4-methylsulfinybutyl glucosinolate,RAA) is one of glucosinolates. Sulforaphane (SF) is the hydrolysis product of glucoraphanin, and it hasbeen proved to be with higher anti-cancer bioactivity. Some studies have suggested that sulforaphanemay reduce the risk of many types of cancers, including liver, stomach, lung, breast, and bladder，and soon, as well as myocardial infarction. Nowadays most of studies on medical researches based onsulforaphane have been widely reported, but there are few studies on germplasm selection with highersulforaphane content in Brassica vegetables, variation of sulforaphane content in different organs ofbroccoli during the whole development stages, genetic analysis and QTL location of sulforaphanecontent in broccoli, and regulation mechanism of sulforaphane gereration related some important genes.
In the study, four varieties of Brassica, which are cabbage, broccoli, Chinese kale and kohlrabi, andtwo populations of double haploid (DH,176) and six generations (P1, P2, F1, B1, B2and F2) of broccoliwere chosen. This study focused on HPLC (High Performance Liquid Chromatography) analysis ofedibile and the other part organs of four varieties of Brassica, variation of sulforaphane content indifferent organs of broccoli during the whole development stages, genetic analysis of sulforaphanecontent in florets of broccoli based on populations of DH and six generations, construction of geneticlinkage map based on DH population of brcooli, QTL location of sulforaphane content in florets ofbroccoli, and RT-PCR (Reverse transcription-PCR) analysis of genes cotrolling sulforahane generation,at the same time one RNAi expression vector (AOP2) and another35S expression promoter vector(CYP79F1) were designed successfully.
The central results of this study were following:
(1) Reverse phase high performance liquid chromatography (RP-HPLC) and ultra-performance liquidchromatography tandem mass spectrum (UPLC-MS/MS) were used for determination and identificationthe composition of sulforaphane, at the same time orgthogonal design L9(34) was also used foroptimizing the extract system of sulforaphane from broccoli. The result suggested that there was a goodstandard curve Y=3.69e-004X-1.26(R2=0.9994) based on gradient program with good precision(RSD=2.50e-9%, n=6) and recovery96.2%(n=6). UPLC-MS/MS showed that retention time (2.17) andmolecular weight (177.29) of extract sample were consistent with the standard sample.
(2) HPLC analysis of sulforaphane contents in Brasscica vegetables: edible part of48lines of inbredand F1hybrid of cabbage; florets, young stems and leaves of29inbred lines of broccoli; edible part of44lines of Chinese kale (Inbred line and F1hybrid); edile part of39lines of kohlrabi (Inbred line and F1hybrid). The result suggested that the highest content of sulforaphane was in florets of broccoli, theothers from high to low were separately edible parts of Chinese kale, kohlrabi and cabbage. Thedistributions of sulforaphane content from higher to the lowest were individually floret, stem and leaf.Finally forteen lines with higher content of sulforaphane in four varieties were obtained in the study.
(3) Four lines of broccoli, including two inbred lines of B691and B692, two hybrids F1of B693and B694crossed from B692×B691and B691×B692, were chosen for study on variation of suforaphanecontent in different organs of broccoli during different development stages. The study firstly revealedthe different variances of sulforaphane content in the whole development stages of broccoli, and therewere higher content in organs of ripe seed, buds at bolting, leaves twenty days after planted in the fieldand seedling in early stages of sprouting.
(4) One DH population of broccoli from F1(86101×90196) crossed from two inbred lines withsignificant difference in sulforaphane content. The DH population contained176lines constructed bymicrospore cultivation, and one population of six generation (P1, P2, F1, B1, B2and F2) was alsoconstructed from the same parents with DH population. The content of sulforaphane was detected byhigh performance liquid chromatography (HPLC), and plant mixed major gene plus polygeneinheritance model was used for genetic analysis of sulforaphane content in floret of broccoli in twopopulations. The result suggested that the quantitative trait of sulforaphane content was controlled bythree additive-epitasis major gene plus polygene (G-1) in DH population, and the major geneheritability was89.28%, while polygene heritability was2.58%, major gene playing a key role incontrolling sulforaphane content. The model was used for analysis of six generation, and the resultsuggested that sulforaphane content was controlled by two major genes with additive dominant epitasiseffect plus polygene with additive dominant epitasis effect (E-1-0). And major gene heritability andpolygene heritability were both nearly50%, which suggested that the quantitative trait was controlledby major gene and polygene together.
(5) One DH population (176) of broccoli from F1(86101×90196) was used for construction linkagemap.314pairs of SSR primers selected from3786pairs of primers (EST-SSR and gSSR) with goodpolymorphism, and they were repeatable, difference and stable. The software of Joinmap4.0wasapplied for construction of the linkage map, the linkage map covered1293.32cM of the genomecontaing268pairs of SSR primers, and all the primers distributed in9linkage groups. In the linkagemap, the longest linkage group was LG1covering302.8cM of the genome, and the shortest was LG7covering26.9cM, both of them contained respectively60and16pairs of SSR primers, the averagegenetic distance was respectively5.1cM and1.7cM. At the same time, the nearest genetic distance ofprimers was also LG7.
(6) Statistic software of SPSS10.0and MapQTL4.0software were used for QTL mapping ofsulforaphane contents in floret of broccoli based on DH populaton (176). The result suggested that atotal of13QTL were tested by interval mapping (IM) and multiple QTL modes (MQM) together, andthey distributed in four linkage groups (LG1, LG6, LG8and LG9) individually with3,2,1and7QTL.
(7) The gene of AOP2was cloned from broccoli (B108) by nest PCR amplification, and then it wasinserted into pSURE-T vector. The cloned fragment of1696bp was validated by white test, restrictionenzyme and sequencing. The sequence performanced in NCBI by analysis of Blastx, finally15fragments above73%similarity and14fragments above95%similarity were obtained. Most of thefragments by Blastx analysis belong to Brassica crops. Phylogenetic trees of MP and NJ wereconstructed based on similar sequences of AOP2by MEGA, and different copies were found in Brassica crops, suggesting the gene AOP2was with higher diversity. At the same time, it had been alsoscanned in the genome of cabbage and Chinese cabbage by Blastx and location on chromosome. In thestudy, there were respectively three copies with different similarities found in chromosome of cabbageand Chinse cabbage, and three copies distributed in chromosome3, chromosome8and chromosome9of cabbage, while another three copies were in chromosome2, chromosome3and chromosome9ofChinese cabbage. And the similarity of all the copies was above90%.
(8) Broccoli B108was used for analysis of correlation between sulforaphane content and theexpression levels of the genes of CYP79F1and AOP2. CYP79F1and AOP2were separately related withmechanism of aliphatic gulcosinolate. Kinds of flower organs, including floret in commercial stage, topbud, bud, bud one day before flowering, flower at bolting, and ripe seed, were detected by RP-HPLC,and reverse transcription PCR (RT-PCR) was used for analysis of CYP79F1and AOP2in related organsduring different period. The data revealed that there was significant difference of sulforaphane contentsat5%during all stages. Reverse transcription PCR (RT-PCR) suggested that there were significantdifferences at1%level of AOP2and CYP79F1, and both of the genes expressed in flower higher thanthe other stages. Correlation analysis of the expression levels of the genes AOP2and CYP79F1suggested that positive correlation happened in the process. But negative correlation was found betweenthe content of sulforaphane and the expression amount of the genes AOP2and CYP79F1.
(9) RNAi and35S promoter of the genes AOP2and CYP79F1were designed in the study. Theexpression vector of pJG081-AOP2showed Hyg resistant in plant and Kan resistant in bacteria. Twoexpression vectors of CYP79F1both contained EGFP reporter.
引文
1.陈琛，庄木，李康宁，刘玉梅，杨丽梅，张扬勇，程斐，孙培田，方智远．甘蓝EST-SSR标记的开发与应用．园艺学报，2010，37（2）：221~228.
2.陈书霞，房玉林，王晓武，程智慧，方智远，孙培田.甘蓝类作物抽薹期及开花期数量性状的研究进展.中国农学通报，2005，21（7）：298~301.
3.陈新娟，朱祝军，杨静，刘永华.芥蓝叶和薹的硫代葡萄糖苷组分及含量.园艺学报，2006，33（4）：741~744.
4.方智远，孙培田.甘蓝几个数量性状遗传力研究初报.中国蔬菜，1981，1：23~25.
5.方智远，刘玉梅，杨丽梅等.我国甘蓝遗传育种研究概况.园艺学报，2002，29（增刊)：657~663.
6.盖钧镒，章元明，王建康.植物数量性状遗传体系.北京：科学出版社，2003.
7.盖钧镒，章元明，王健康. QTL混合遗传模型扩展至2对主基因+多基因时的多世代联合分析.作物学报，2000，26（4）：385~391.
8.郭永泽，绍辉，张玉婷，刘磊，李辉，宋淑荣. GC-MS/MS法测定油菜籽中异丙甲草胺残留量.农药，2009，48（10）：742~743.
9.韩建明，侯喜林，史公军，陈沁滨.不结球白菜株高性状主基因+多基因遗传分析.南京农业大学学报，2008，31（1）：23~26.
10.胡学军，邹国林.甘蓝分子连锁图的构建与品质性状的QTL定位.武汉植物学研究，2004，22（6）：482~485.
11.腊贵晓，方萍，李亚娟，王月.液相色谱质谱联用分离、鉴定芥蓝中脱硫芥子油苷.2008，34（5）：557~563.
12.梁浩，袁其朋，东惠茹，钱忠明，刘玉梅.十字花科植物种子中莱菔硫烷含量的比较.中国药学杂志，2004，39（12）：898~899.
13.廖永翠，宋明，王辉，徐东辉，王晓武.大白菜中硫代葡萄糖苷的鉴定及含量分析.园艺学报，2011，38（5）：936~969.
14.何洪巨，陈杭，Schnitzler W H.芸薹属蔬菜中硫代葡萄糖苷鉴定与含量分析.中国农业科学，2002，35（2）：192~197.
15.姜长鉴，莫惠栋.质量-数量性状的遗传分析Ⅳ-极大似然法的应用.作物学报，1995，21（6）：641~648.
16.刘二艳.青花菜花球外观品质性状的遗传分析和分子标记研究[硕士学位论文].北京：中国农业科学院，2009.
17.刘二艳，刘玉梅，方智远，杨丽梅，庄木，张扬勇，袁素霞，孙继峰，李占省，孙培田.青花菜花球‘荚叶’性状主基因+多基因遗传分析.园艺学报，2009，36（11）：1611~1618.
18.李纪锁，沈火林，石正强.鲜食番茄果实中番茄红素含量的主基因-多基因混合遗传分析.遗传，2006，28（4）：458~462.
19.李余生，朱镇，张亚东，赵凌，王才林.水稻稻曲病抗性的主基因+多基因混合遗传模型分析.作物学报，2008，34（10）：1728~1733.
20.李占省，刘玉梅，方智远，杨丽梅，庄木，张扬勇，孙培田.甘蓝叶球中莱菔硫烷含量的检测与分析[A].中国十字花科蔬菜研究进展，2009.
21.李占省，刘玉梅，方智远，杨丽梅，庄木，张扬勇，孙培田，靳松.青花菜不同营养器官中莱菔硫烷含量的HPLC检测与分析.园艺学报，2009，36（S）：1971.
22.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.
23.马育华.植物育种的数量遗传学基础.南京：江苏科学技术出版社，1982.
24.缪体云，刘玉梅，方智远，杨丽梅，庄木，张扬勇，袁素霞，孙培田.一个结球甘蓝DH群体主要农艺性状的遗传效应分析.园艺学报，2008，35（1）：59~64.
25.钱丽丽，刘江丽，李扬.西兰花中硫代葡萄糖苷的提取及抑菌试验初报.中国农学通报，2008，24：335~338.
26.邱海荣，姚雪琴，谢祝捷，施元吉，乔永进，吴震.反相高效液相色谱法检测青花菜和花椰菜中4-甲基亚磺酰丁基硫苷.上海农业学报，2008，24（4）：72~74.
27.任丽娟，颜伟，陈怀谷，马鸿翔.小麦纹枯病抗性的主基因+多基因遗传分析.江苏农业学报，2010，1156~1161.
28.沈莲清，苏光耀，王奎武.西兰花种子中硫甙酶解产物萝卜硫素的提纯与抗肿瘤的体外试验研究.中国食品学报，2008，8（5）：15~20.
29.孙勃，许映君，徐铁锋，袁高峰，郭容芳，汪炳良，汪俏梅.青花菜不同器官生物活性物质和营养成分的研究.园艺学报，2010，37（1）：59~64.
30.徐东辉.白菜类作物硫代葡萄糖甙及一些主要谢组分的遗传分析[博士学位论文].北京：中国农业科学院，2007.
31.王辉，孙日飞，邓杰，武剑，王晓武.控制白菜3-丁烯基硫代葡萄糖苷积累的QTL定位及分析.园艺学报，2011，38（7）：1283~1290.
32.王健康.数量性状主基因-多基因混合遗传模型的鉴别和遗传参数估计的研究[博士学位论文].南京：南京农业大学，1996.
33.王丽红，刘娟，王桂艳，杜娟.光果葶苈中莱菔硫烷的提取及含量测定.中药与天然药，2010，27（4）：316~319.
34.王晓武，方智远，孙培田.甘蓝显性雄性不育基因的延长随机引物扩增DNA（ERPAD）标记.园艺学报，1999，26（1）：23~27.
35.王晓武，娄平，何杭军，杨宝军，张延国，赵建军.利用芥蓝×青花菜DH群体构建AFLP连锁图谱.园艺学报，2005，32（1）：30~34.
36.吴元峰，沈莲清，毛建卫，黄光荣，袁海娜.芸薹属植物种子中萝卜硫素的提取工艺研究.食品与生物技术学报，2009，28（5）：647~651.
37.夏薇，赵秀娟，吴坤，苑林宏.北方12种蔬菜中莱菔硫烷含量的测定.疾病控制杂志，2005，9（3）：209~211.
38.谢祝捷，李媛，姚雪琴，邱海荣.青花菜基因型和环境互作对花球4-甲基亚磺酰丁基硫苷含量的影响.园艺学报，2010，37（4）：625~630.
39.杨丽梅，方智远，刘玉梅，庄木，张扬勇，孙培田.“十一五”我国甘蓝遗传育种研究进展.中国蔬菜，2011，10（2）：1~11.
40.严慧玲，方智远，刘玉梅，王永建，杨丽梅，庄木，张扬勇，孙培田.甘蓝显性雄性不育材料DGMS79-399-3不育性的遗传效应分析.园艺学报，2007，34（1）：93~98.
41.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.
42.张婵娟，郭晓玲，孟青，冯毅凡.不同品种西兰花种子中莱菔硫烷的HPLC分析.广东药学院学报，2007，23（5）：505~508.
43.张国良，赵秀娟，张宇秋，赵艳，张海金，吴坤.蔬菜中莱菔硫烷提取工艺的正交优选.哈尔滨医科大学学报，2009，43（6）：626~628.
44.张培通，朱协飞，郭旺珍，俞敬忠，张天真.高产棉花品种泗棉3号产量及其产量构成因素的遗传分析.作物学报，2006，32（17）：1011~1017.
45.朱定祥，凤权.十字花科蔬菜西兰花中莱菔硫烷含量的研究.天然产物研究与开发，2009，5：135~137.
46.朱军.运用混合线性模型定位复杂数量性状基因的方法.浙江大学学报（自然科学版），1999，33（3）：327~335.
47.朱穗琴，张宪银，薛庆中.烟草两个DH群体农艺性状的遗传分析.浙江大学学报：农业与生命科学版，2004，30（5）：477~481.
48. Abercrombie JM, Farnham MW, Rushing JW. Genetic combining ability of glucoraphanin leveland other horticultural traits of broccoli. Euphytica.2005,143:145-151.
49. Adamska E, Cegielska-Taras T, Kaczmarek Z, Szala L. Multivariate approach to evaluating thefatty acid composition of seed oil in a doubled haploid population of winter oilseed rape (Brassicanapus L.). J Appl Genet.2004,45(4):419-425.
50. Agerbirk N, Olsen CE, Chew FS, Orgaard M. Variable glucosinolate profiles of Cardaminepratensis (Brassicaceae) with equal chromosome numbers. J Agric Food Chem.2010,58(8):4693-4700.
51. Agrawal S, Winnik B, Buckley B, Mi L, Chung FL, Cook TJ. Simultaneous determination ofsulforaphane and its major metabolites from biological matrices with liquidchromatography-tandem mass spectroscopy. J Chromatogr B Analyt Technol Biomed Life Sci.2006,840(2):99-107.
52. Agrawal S, Winnik B, Buckley B, Mi LX, Chung FL, Cook TJ. Simultaneous determination ofsulforaphane and its major metabolites from biological matrices with liquidchromatography-tandem mass spectroscopy. J Chromatogr B.2006,840(2):99-107.
53. Ahn YH, Hwang Y, Liu H, Wang XJ, Zhang Y, Stephenson KK,. Electrophilic tuning of thechemoprotective natural product sulforaphane. Proc Natl Acad Sci U S A.2010,107(21):9590-9595.
54. Al Janobi AA, Mithen RF, Gasper AV, Shaw PN, Middleton RJ, Ortori CA. Quantitativemeasurement of sulforaphane, iberin and their mercapturic acid pathway metabolites in humanplasma and urine using liquid chromatography-tandem electrospray ionisation mass spectrometry. JChromatogr B Analyt Technol Biomed Life Sci.2006,844(2):223-234.
55. Andelova H, Rudolf E, Cervinka M. In vitro antiproliferative effects of sulforaphane on humancolon cancer cell line SW620. Acta Medica (Hradec Kralove).2007,50(3):171-176.
56. Angeloni C, Leoncini E, Malaguti M, Angelini S, Hrelia P, Hrelia S. Modulation of phase IIenzymes by sulforaphane: implications for its cardioprotective potential. J Agric Food Chem.2009,57(12):5615-5622.
57. Annabi B, Rojas-Sutterlin S, Laroche M, Lachambre MP, Moumdjian R, Beliveau R. Thediet-derived sulforaphane inhibits matrix metalloproteinase-9-activated human brain microvascularendothelial cell migration and tubulogenesis. Mol Nutr Food Res.2008,52(6):692-700.
58. Aherne FX, Lewis AJ. The nutritive value of faba beans and low glucosinolate rapeseed meal forswine. Adv Exp Med Biol.1978,105:453-471.
59. Aires A, Mota VR, Saavedra MJ, Monteiro AA, Simoes M, Rosa EA,. Initial in vitro evaluationsof the antibacterial activities of glucosinolate enzymatic hydrolysis products against plantpathogenic bacteria. J Appl Microbiol.2009,106(6):2096-2105.
60. Al-Gendy AA, Lockwood GB. Production of glucosinolate hydrolysis products in Farsetia aegyptiasuspension cultures following elicitation. Fitoterapia.2005,76(3-4):288-295.
61. Azizi SN, Amiri-Besheli B, Sharifi-Mehr S. The Isolation and Determination of Sulforaphane fromBroccoli Tissues by Reverse Phase-High Performance Liquid Chromatography. J Chin ChemSoc-Taip.2011,58(7):906-910.
62. Bak S, Nielsen HL, Halkier BA. The presence of CYP79homologues in glucosinolate-producingplants shows evolutionary conservation of the enzymes in the conversion of amino acid toaldoxime in the biosynthesis of cyanogenic glucosides and glucosinolates. Plant Mol Biol.1998,38(5):725-734.
63. Bednarek P, Pislewska-Bednarek M, Svatos A, Schneider B, Doubsky J, Mansurova M,. Aglucosinolate metabolism pathway in living plant cells mediates broad-spectrum antifungal defense.Science.2009,323(5910):101-106.
64. Bennett RN, Hick AJ, Dawson GW, Wallsgrove RM. Glucosinolate Biosynthesis (FurtherCharacterization of the Aldoxime-Forming Microsomal Monooxygenases in Oilseed Rape Leaves).Plant Physiol.1995,109(1):299-305.
65. Bhaskar B, Ahuja I, Janeja S, Banga S. Intergeneric hybridization between Erucastrum canarienseand Brassica rapa. Genetic relatedness between E(C) and A genomes. Theor Appl Genet.2002,105(5):754-758.
66. Bohuon EJR. Alignment of the conserved C genomes of Brassica oleracea and Brassica napus.TAG1996,93(5/6):833-839.
67. Brown PD, Tokuhisa JG, Reichelt M, Gershenzon J. Variation of glucosinolate accumulationamong different organs and developmental stages of Arabidopsis thaliana. Phytochemistry.2003,62(3):471-481.
68. Brader G, Mikkelsen MD, Halkier BA, Tapio Palva E. Altering glucosinolate profiles modulatesdisease resistance in plants. Plant J.2006,46(5):758-767.
69. Bressan M, Roncato MA, Bellvert F, Comte G, Haichar FZ, Achouak W. Exogenous glucosinolateproduced by Arabidopsis thaliana has an impact on microbes in the rhizosphere and plant roots.ISME J.2009,3(11):1243-1257.
70. Broekaert N, Van Peteghem C, Daeseleire E, Sticker D, Van Poucke C. Development andvalidation of an UPLC-MS/MS method for the determination of ionophoric and syntheticcoccidiostats in vegetables. Analytical and Bioanalytical Chemistry.2011,401(10):3335-3344.
71. Bridges M, Jones AM, Bones AM, Hodgson C, Cole R, Bartlet E. Spatial organization of theglucosinolate-myrosinase system in brassica specialist aphids is similar to that of the host plant.Proc Biol Sci.2002,269(1487):187-191.
72. Camargo WY, Champagne G. Mapping of quantitative trait loci controlling resistance of Brassicaoleracea to Xanthomonas campestris PV. Campestris. Phytopathology1995,85(10):1296-1300.
73. Chakrabarty SK, Chandrashekar US, Prasad M, Yadav JB, Singh R, Singh JN. Protogyny andself-incompatibility Indian mustard (Brassica juncea L. Czern&Coss)-a new tool for hybriddevelopment. Indian J Genet Pl Br.2011,71(3):294-298.
74. Clarke JD, Dashwood RH, Ho E. Multi-targeted prevention of cancer by sulforaphane. Cancer Lett.2008,269(2):291-304.
75. Chari VD, Sharma DK, Prasad PSR. Methane hydrate phase stability with lower mole fractions oftetrahydrofuran (THF) and tert-butylamine (t-BuNH2). Fluid Phase Equilibr.2012,315:126-130.
76. Chen S, Glawischnig E, Jorgensen K, Naur P, Jorgensen B, Olsen CE. CYP79F1and CYP79F2have distinct functions in the biosynthesis of aliphatic glucosinolates in Arabidopsis. Plant J.2003,33(5):923-937.
77. Chen J, Song L, Dai J, Gan N, Liu Z. Effects of microcystins on the growth and the activity ofsuperoxide dismutase and peroxidase of rape (Brassica napus L.) and rice (Oryza sativa L.).Toxicon.2004,43(4):393-400.
78. Cheung WY. Comparison of the genetic maps of Brassica napus and Brassica oleracea. TAG.1996,94:569-582.
79. Chiang WCK, Pusateri DJ, Leitz REA. Gas chromatography mass spectrometry method for thedetermination of sulforaphane and sulforaphane nitrile in broccoli. J Agri Food Chem.1998,46(3):1018-1021.
80. Chyi YS. A genetic linkage map of restriction fragment length polymorphism loci for Brassica rapa(syn. Campestris). J Genome.1992,35:746-757.
81. Cottaz S, Henrissat B, Driguez H. Mechanism-based inhibition and stereochemistry ofglucosinolate hydrolysis by myrosinase. Biochemistry.1996,35(48):15256-15259.
82. Cramer JM, Teran-Garcia M, Jeffery EH. Enhancing sulforaphane absorption and excretion inhealthy men through the combined consumption of fresh broccoli sprouts and a glucoraphanin-richpowder. Br J Nutr.2011:1-6.
83. Cramer JM, Jeffery EH. Sulforaphane absorption and excretion following ingestion of asemi-purified broccoli powder rich in glucoraphanin and broccoli sprouts in healthy men. NutrCancer.2011,63(2):196-201.
84. David WA, Gardiner BO. Persistence of a granulosis virus of Pieris brassicae on cabbage leaves. JInvertebr Pathol.1966,8(2):180-183.
85. Daxenbichler ME, VanEtten CH. Glucosinolates and derived products in cruciferous vegetables:gas-liquid chromatographic determination of the aglucon derivatives from cabbage. J Assoc ofAnal Chem.1977,60(4):950-953.
86. Dinkova-Kostova AT, Fahey JW, Benedict AL, Jenkins SN, Ye L, Wehage SL. Dietaryglucoraphanin-rich broccoli sprout extracts protect against UV radiation-induced skincarcinogenesis in SKH-1hairless mice. Photochem Photobiol Sci.2010,9(4):597-600.
87. Dinkova-Kostova AT, Jenkins SN, Fahey JW, Ye L, Wehage SL, Liby KT. Protection againstUV-light-induced skin carcinogenesis in SKH-1high-risk mice by sulforaphane-containingbroccoli sprout extracts. Cancer Lett.2006,240(2):243-252.
88. Elikind Y and Cahaner A. A mixed model for the effects of single gene，polygenes and theirinteraction on quantitative traits. The model and experimental design. Theor Appl Genet.1986,72:377-383.
89. Elston RC and Stewart. The analysis of quantitative traits for simple genetic models from parental，F1and bachcross data. Genetic.1973,73:695-711.
90. Farnham MW, Stephenson KK, Fahey JW. Glucoraphanin level in broccoli seed is largelydetermined by genotype. HortScience.2005,40(1):50-53.
91. Farnham MW, Wilson PE, Stephenson KK, Fahey JW. Genetic and environmental effects onglucosinolate content and chemoprotective potency of broccoli. Plant Breeding.2004,123(1):60-65.
92. Feng J, Long Y, Shi L, Shi JQ, Barker G, Meng JL. Characterization of metabolite quantitative traitloci and metabolic networks that control glucosinolate concentration in the seeds and leaves ofBrassica napus. New Phytologist.2012,193(1):96-108.
93. Federico L, Inigurez L, Amy VV, Tomas CO. Development of a set of pubili SSR markers deribedfrom genomic sequence of a rapid cycling Brassica oleracea L. genotype. TAG.2008,117:977-985.
94. Field B, Furniss C, Wilkinson A, Mithen R. Expression of a Brassica isopropylmalate synthasegene in Arabidopsis perturbs both glucosinolate and amino acid metabolism. Plant Mol Biol.2006,60(5):717-727.
95. Frydoonfar HR, McGrath DR, Spigelman AD. The effect of indole-3-carbinol and sulforaphane ona prostate cancer cell line. ANZ J Surg.2003,73(3):154-156.
96. Gao M, Li G, Potter D, McCombie WR, Quiros CF. Comparative analysis of methylthioalkylmalatesynthase (MAM) gene family and flanking DNA sequences in Brassica oleracea and Arabidopsisthaliana. Plant Cell Rep.2006,25(6):592-598.
97. Galan MV, Kishan AA, Silverman AL. Oral broccoli sprouts for the treatment of Helicobacterpylori infection: a preliminary report. Dig Dis Sci.2004,49(7-8):1088-1090.
98. Gergely A, Horvath P, Szasz G, Veress G. Gradient HPLC separation of dehydroepiandrosterone(DHEA) from its metabolites and biological congeners: role of tetrahydrofuran in thechromatographic mechanism. Analytical and Bioanalytical Chemistry.2009,394(8):2105-2109.
99. Gigolashvili T, Engqvist M, Yatusevich R, Muller C, Flugge UI. HAG2/MYB76andHAG3/MYB29exert a specific and coordinated control on the regulation of aliphatic glucosinolatebiosynthesis in Arabidopsis thaliana. New Phytol.2008,177(3):627-642.
100. Gorman PD, English NJ, MacElroy JMD. Dynamical cage behaviour and hydrogen migration inhydrogen and hydrogen-tetrahydrofuran clathrate hydrates. J Chem Phys.2012,136(4):044-506.
101. Gorissen A, Kraut NU, de Visser R, de Vries M, Roelofsen H, Vonk RJ. No de novo sulforaphanebiosynthesis in broccoli seedlings. Food Chemistry.2011,127(1):192-196.
102. Grubb CD, Abel S. Glucosinolate metabolism and its control. Trends Plant Sci.2006,11(2):89-100.
103. Hale AL, Farnham MW, Nzaramba MN, Kimbeng CA. Heterosis for horticultural traits in broccoli.Theor Appl Genet.2007,115(3):351-360.
104. Hansen CH, Du L, Naur P, Olsen CE, Axelsen KB, Hick AJ. CYP83b1is the oxime-metabolizingenzyme in the glucosinolate pathway in Arabidopsis. J Biol Chem.2001,276(27):24790-24796.
105. Heidel AJ, Clauss MJ, Kroymann J, Savolainen O, Mitchell-Olds T. Natural variation in MAMwithin and between populations of Arabidopsis lyrata determines glucosinolate phenotype.Genetics.2006,173(3):1629-1636.
106. Hu R, Xu C, Shen G, Jain MR, Khor TO, Gopalkrishnan A. Gene expression profiles induced bycancer chemopreventive isothiocyanate sulforaphane in the liver of C57BL/6J mice andC57BL/6J/Nrf2(-/-) mice. Cancer Lett.2006,243(2):170-192.
107. Iori R, Bernardi R, Gueyrard D, Rollin P, Palmieri S. Formation of glucoraphanin bychemoselective oxidation of natural glucoerucin: a chemoenzymatic route to sulforaphane.Bioorg Med Chem Lett.1999,9(7):1047-1048.
108. Kennard WC, Slocum MK, Figdore SS, Osborn TC. Genetic analysis of morphological variation inBrassica oleracea using molecular markers. TAG.1994,87:721-732.
109. Kianian SF and Quiros CF. Generation of a Brassica oleracea composite RFLP map: linkagearrangement among various population and evolutionary implications. TAG.1992,84:544-554.
110. Kliebenstein DJ, Lambrix VM, Reichelt M, Gershenzon J, Mitchell-Olds T. Gene duplication in thediversification of secondary metabolism: tandem2-oxoglutarate-dependent dioxygenases controlglucosinolate biosynthesis in Arabidopsis. Plant Cell.2001,13(3):681-693.
111. Kliebenstein DJ, Kroymann J, Brown P, Figuth A, Pedersen D, Gershenzon J. Genetic control ofnatural variation in Arabidopsis glucosinolate accumulation. Plant Physiol.2001,126(2):811-25.
112. Kliebenstein DJ, Kroymann J, Mitchell-Olds T. The glucosinolate-myrosinase system in anecological and evolutionary context. Curr Opin Plant Biol.2005,8(3):264-71.
113. Kramer CC, Polewicz H, Osborn TC. Evaluation of QTL alleles from exotic sources for hybridseed yield in the original and different genetic backgrounds of spring-type Brassica napus L. MolBreeding.2009,24(4):419-431.
114. Krajka-Kuzniak V, Szaefer H, Bartoszek A, Baer-Dubowska W. Modulation of rat hepatic andkidney phase II enzymes by cabbage juices: comparison with the effects of indole-3-carbinol andphenethyl isothiocyanate. Br J Nutr.2011,105(6):816-826.
115. Larder E S, Holstein S. Mapping Mendetien factors underlying quaatitative traits using RFLPlinkage maps. Genetics.1989,121:185-199.
116. Landry BS, Hubert N, Crete R, Chang MS, Lincoln SE, and Etho T. A genetic map of Brassicaoleracea based on RFLP markers detected with expressed DNA sequences and mapping ofresistance genes to race2of Plasmodiophora brassicae (Woronin). Genome.1992,35:409-420.
117. Leveques A, Actis-Goretta L, Rein MJ, Williamson G, Dionisi F, Giuffrida F. UPLC-MS/MSquantification of total hesperetin and hesperetin enantiomers in biological matrices. J PharmaceutBiomed.2012,57:1-6.
118. Li CF, Liang H, Yuan QP, Hou XD. Optimization of sulforaphane separation from broccoli seedsby macroporous resins. Separ Sci Technol.2008,43(3):609-623.
119. Liang H, Yuan QP, Dong HR, Liu YM. Determination of sulforaphane in broccoli and cabbage byhigh-performance liquid chromatography. J Food Compos Anal.2006,19(5):473-476.
120. Liang H, Yuan Q, Liu M. Simultaneous determination of glucoraphanin and sulforaphane inBrassica oleracea seeds by high-performance liquid chromatography with evaporativelight-scattering detector. Nat Prod Res.2012,1:1-4.
121. Liang H, Yuan QP, Xiao Q. Effects of metal ions on myrosinase activity and the formation ofsulforaphane in broccoli seed. J Mol Catal B-Enzym.2006,43(4):19-22.
122. Lou P，Kang JG，Zhang GY，Guusje B，Fang ZY，Wang XW． Transcript profiling of a dominantmale sterile mutan（tMs-cd1）in cabbage during flower bud development．Plant Science.2007,172：111-119.
123. Magrath R, Bano F, Morgner M, Parkin I, Sharpe A, Lister C, Dean C, Turner J, Lydiate D, MithenR. Genetics of aliphatic glucosinolates.1. side-chain elongation in Brassica napus and Arabidopsisthaliana. Heredity.1994,72:290-299.
124. Matusheski NV, Jeffery EH. Comparison of the bioactivity of two glucoraphanin hydrolysisproducts found in broccoli, sulforaphane and sulforaphane nitrile. J Agric Food Chem.2001,49(12):5743-5749.
125. Mewis I, Tokuhisa JG, Schultz JC, Appel HM, Ulrichs C, Gershenzon J. Gene expression andglucosinolate accumulation in Arabidopsis thaliana in response to generalist and specialistherbivores of different feeding guilds and the role of defense signaling pathways. Phytochemistry.2006,67(22):2450-62.
126. Mikkelsen MD, Petersen BL, Glawischnig E, Jensen AB, Andreasson E, Halkier BA. Moriguchi K.A genetic map based on RAPD, RFLP, isozyme, morphological markers and QTL analysis forclubroot resistance in Brassica oleracea J. Breeding Science.1999,49(4):257-265.
127. Mithen R, Clarke J, Lister C, Dean C. Genetics of aliphatic glucosinolates. III. Side chain structureof aliphatic glucosinolates in Arabidopsis thaliana. Heredity.1995,74:210-215.
128. Mikkelsen MD, petersen BL, Glawischnig E, Jensen AB, Andreasson E, Halkier BA. Modulationof CYP79genes and glucosinolate profiles in Arabidopsis by defense signaling pathways. PlantPhysiol.2003,131(1):298-308.
129. Morton NE and MacLean CJ. Analysis of family resemblance. III. Complex segregation ofquantitative traits. Am J Hum Genet.1974,26:489-503.
130. Mukherjee S, Gangopadhyay H, Das DK. Broccoli: a unique vegetable that protects mammalianhearts through the redox cycling of the thioredoxin superfamily. J Agric Food Chem.2008,56(2):609-617.
131. Nakagawa K, Umeda T, Higuchi O, Tsuzuki T, Suzuki T, Miyazawa T. Evaporative light-scatteringanalysis of sulforaphane in broccoli samples: Quality of broccoli products regarding sulforaphanecontents. J Agric Food Chem.2006,54(7):2479-2483.
132. Neal CS, Fredericks DP, Griffiths CA, Neale AD. The characterisation of AOP2: a gene associatedwith the biosynthesis of aliphatic alkenyl glucosinolates in Arabidopsis thaliana. BMC Plant Biol.2010,10:170.
133. Nho CW, Jeffery E. The synergistic upregulation of phase II detoxification enzymes byglucosinolate breakdown products in cruciferous vegetables. Toxicol Appl Pharmacol.2001,174(2):146-152.
134. Osborn TC, Kole C, Parkin IAP. Comparison of flowering time genes in Brassica rapa, B. napusand Arabidopsis thaliana. Genetics1997,156:1123-1129.
135. Piao CS, Gao S, Lee GH, Kim do S, Park BH, Chae SW. Sulforaphane protects ischemic injury ofhearts through antioxidant pathway and mitochondrial K (ATP) channels. Pharmacol Res.2010,61(4):342-348.
136. Pradhan A, Plummer JA, Nelson MN, Cowling WA, Yan GJ. Successful induction of trigenomichexaploid Brassica from a triploid hybrid of B. napus L. and B. nigra (L.) Koch. Euphytica.2010,176(1):87-98.
137. Ramchiary N, Bisht NC, Gupta V, Mukhopadhyay A, Arumugam N, Sodhi YS. QTL analysisreveals context-dependent loci for seed glucosinolate trait in the oilseed Brassica juncea:importance of recurrent selection backcross scheme for the identification of 'true' QTL. Theor ApplGenet.2007,116(1):77-85.
138. Rangkadilok N, Nicolas ME, Bennett RN, Eagling DR, Premier RR, Taylor PW. The effect ofsulfur fertilizer on glucoraphanin levels in broccoli (B. oleracea L. var. italica) at different growthstages. J Agric Food Chem.2004,52(9):2632-9.
139. Reintanz B, Lehnen M, Reichelt M, Gershenzon J, Kowalczyk M, Sandberg G. Bus, a bushyArabidopsis CYP79F1knockout mutant with abolished synthesis of short-chain aliphaticglucosinolates. Plant Cell.2001,13(2):351-367.
140. Resta D, Brambilla F, Arnoldi A. HPLC-Chip-Multiple Reaction Monitoring (MRM) method forthe label-free absolute quantification of gamma-conglutin in lupin: Proteotypic peptides andstandard addition method. Food Chemistry.2012,131(1):126-133.
141. Rose CW, Millwood RJ, Moon HS, Rao MR, Halfhill MD, Raymer PL. Genetic load andtransgenic mitigating genes in transgenic Brassica rapa (field mustard) x Brassica napus (oilseedrape) hybrid populations. Bmc Biotechnology.2009,9.
142. Ritz SA, Wan J, Diaz-Sanchez D. Sulforaphane-stimulated phase II enzyme induction inhibitscytokine production by airway epithelial cells stimulated with diesel extract. Am J Physiol LungCell Mol Physiol.2007,292(1): L33-39.
143. Sebastian RL, Kearsey MJ, King GJ. Identification of quantitative trait loci controllingdevelopmental characteristics of Brassica oleracea L. Theor Appl Genet.2002,104(4):60l-609.
144. Singh BK, Sharma SR, Singh B. Heterosis for superoxide dismutase, peroxidase and catalaseenzymes in the head of single cross-hybrids of cabbage (Brassica oleracea var. capitata). J Genet.2010,89(2):217-221.
145. Sivakumar G, Aliboni A, Bacchetta L. HPLC screening of anti-cancer sulforaphane from importantEuropean Brassica species. Food Chemistry.2007,104:1761-1764.
146. Slocum M K. Linkage arrangement of restriction length polymorphism loci in Brassica oleracea.TAG1990,80:57-64.
147. Solowiej E, Kasprzycka-Guttman T, Fiedor P, Rowinski W. Chemoprevention ofcancerogenesis--the role of sulforaphane. Acta Pol Pharm.2003,60(1):97-100.
148. Solowiej E, Solowiej J, Godlewski M, Motyl T, Perkowska-Ptasinska A, Jaskiewicz K. Applicationof sulforaphane: histopathological study of intraportal transplanted pancreatic islets into livers ofdiabetic rats. Transplant Proc.2006,38(1):282-283.
149. Solowiej E, Solowiej J, Kasprzycka-Guttman T, Rowinski W. Application of sulforaphane--does itlead to improvement of islet graft survival after warm and/or cold ischemia. Ann Transplant.2004,9(3):68-71.
150. Song N, Osborn TC, Williams PH. Brassica taxonomy based on nuclear restriction fragmentlengthpolymorphism (RFLPs). I. Genome evolution of diploid and amphidiploid species J. Theor ApplGenet.1988,75:784-794.
151. Staack R, Kingston S, Wallig MA, Jeffery EH. A comparison of the individual and collectiveeffects of four glucosinolate breakdown products from brussels sprouts on induction ofdetoxification enzymes. Toxicol Appl Pharmacol.1998,149(1):17-23.
152. Tang SX, Zhuang JJ, Wen YX. Identificat ion of introgressed segments confers ring diseaseresistance in a tetrageneric hybrid of Triticum, Secale, Thinopyrum and Avena. Genome.1997,40:99-103.
153. Tantikanjana T, Mikkelsen MD, Hussain M, Halkier BA, Sundaresan V. Functional analysis of thetandem-duplicated P450genes SPS/BUS/CYP79F1and CYP79F2in glucosinolate biosynthesisand plant development by Ds transposition-generated double mutants. Plant Physiol.2004,135(2):840-848.
154. Thejass P, Kuttan G. Augmentation of natural killer cell and antibody-dependent cellularcytotoxicity in BALB/c mice by sulforaphane, a naturally occurring isothiocyanate from broccolithrough enhanced production of cytokines IL-2and IFN-gamma. ImmunopharmacolImmunotoxicol.2006,28(3):443-457.
155. Voorips RE, Jongerius MC, Kanne HJ. Mapping of two genes for resistance to clubroot(plasmodiophora brassicae) in a population of doubled haploid lines of Brassica oleracea by meansof RFLP and AFLP markers, TAG.1997,94:75-82.
156. Wang H, Lin W, Shen GX, Khor TO, Nomeir AA, Kong AN. Development and Validation of anLC-MS-MS Method for the Simultaneous Determination of Sulforaphane and its Metabolites inRat Plasma and its Application in Pharmacokinetic Studies. J Chromatogr Sci.2011,49(10):801-806.
157. Wang TT, Schoene NW, Milner JA, Kim YS. Broccoli-derived phytochemicals indole-3-carbinoland3,3'-diindolylmethane exerts concentration-dependent pleiotropic effects on prostate cancercells: Comparison with other cancer preventive phytochemicals. Mol Carcinog.2012,51:244-256.
158. Wentzell AM, Rowe HC, Hansen BG, Ticconi C, Halkier BA, Kliebenstein DJ. Linking metabolicQTLs with network and cis-eQTLs controlling biosynthetic pathways. PLoS Genet.2007,3(9):1687-1701.
159. West LG, Meyer KA, Balch BA, Rossi FJ, Schultz MR, Haas GW. Glucoraphanin and4-hydroxyglucobrassicin contents in seeds of59cultivars of broccoli, raab, kohlrabi, radish,cauliflower, brussels sprouts, kale, and cabbage. J Agric Food Chem.2004,52(4):916-926.
160. Wijeyesekera A, Clarke PA, Bictash M, Brown IJ, Fidock M, Ryckmans T. QuantitativeUPLC-MS/MS analysis of the gut microbial co-metabolites phenylacetylglutamine,4-cresylsulphate and hippurate in human urine: INTERMAP Study. Anal Methods-Uk.2012,4(1):65-72.
161. Yabar E, Pedreschi R, Chirinos R, Campos D. Glucosinolate content and myrosinase activityevolution in three maca (Lepidium meyenii Walp.) ecotypes during preharvest, harvest andpostharvest drying. Food Chemistry.2011,127(4):1576-1583.
162. Yan X, Chen S. Regulation of plant glucosinolate metabolism. Planta.2007,226:1343-1352.
163. Yanaka A，Zhang S，Tauchi M. Role of the Nrf2gene in protection and repair of gastric mucosaagainst oxidative stress. Inflammopharmacology.2005,13:83-88.
164. Yu SC, Wang YJ, Zheng XY. Mapping and analysis QTL controlling some morphological traits inChinese cabbage (Brassica campestris L. ssp. pekinensis). Yi Chuan Xue Bao.2003,30(12):1153-1160.
165. Zang YX, Kim JH, Park YD, Kim DH, Hong SB. Metabolic engineering of aliphatic glucosinolatesin Chinese cabbage plants expressing Arabidopsis MAM1, CYP79F1, and CYP83A1. BMB Rep.2008,41(6):472-478.
166. Zeng ZB. Precision mapping of quantitative trait loci. Genetics.1994,136:1457-1468.