| 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.
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