【作者】 李占省；

【导师】 刘玉梅；

【作者基本信息】 中国农业科学院 ， 蔬菜学， 2012， 博士

【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。更多还原

【Abstract】 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.更多还原