| The spatio-temporal expression analysis of genes played an important role in sweetcherry studying. Errors caused by growth environment and genotypes was unavoidable duringpreparation of samples. The stable reference genes should be chosen to normalize theexperimental datas to eliminate the errors.11reference genes had been obtained from sweetcherry and their stability had been evaluated preliminary, but no reports validated theirapplicability had been published.In order to validate the stability and applicability of the selectedreference genes, then obtain more firm result and sift more reliable reference genes forsubsequent studies, the leaves of ‘Tieton’‘Sunburst’‘Lapins’‘Hongdeng’‘Gisaila5’‘Acid blacktavtarian’‘Laiyang short’‘Tianshui xiaoying’and flower organs of ‘Sunburst’ in full-bloomingwere used as materials, qRT-PCR approache was chosen to test the relative expression levels ofthe11candidated reference genes in different genotypes and flower organs of cherry.The phenomenon of malformed fruits caused by adaptability of genotypes and variation ofenvironment happened frequently during the introduction and popularization of sweet cherry.And the situation becomes more and more severly with the enlargement of the cultivated area. Alot of researchers had committed to study this problem, but few of them focused on relatedgenes.In reaction to the phenomenon of malformed fruits in regions unsuitable for sweet cherrydevelopment, the rat of abnormal fruits of ‘Tieton’‘Sunburst’‘Lapins’‘Hongdeng’‘Summit’‘Satohnishiki’ and ‘Regina’ were counted. Meanwhile the flower organs of ‘Tieton’ and‘Sunburst’ whose autumn were different were served as materials, genes related to flower organdevelopment were cloned via slicon cloning technology, and qRT-PCR approache was chosen totest the relative expression levels of the cloned MADS-box genes about flower organdevelopment of sweet cherry. Through all the above methods,we attempted to learn about somekind of generating mechanism of malformed fruits, and relied on the research results to find out the role these genes played in flower organ growth of sweet cherry and provide theoretical basisfor solving the problem of fruits malformation.The acquired results are as follows:1.The geNorm software calculates the gene expression stability measure (M) for a referencegene as the average pairwise variation (V) for that gene with all other tested reference genesanalysis, based on the gene stability measure (M) by geNorm, candidate genes RPL13and RPIIwith the lowest M value were the stable genes.From the variation analysis, V2/3value (0.133)was less than0.15, which suggested two reference genes, RPL13and RPII, were necessary togene expression normalization in leaves of the above tested cherry genotypes.2.In order to validate the effectiveness of reference genes CYP2and TEF2in research ofreproduction-related genes in sweet cherry, Expression of MYB10and CHS genes correlatedwith fruit and flower anthocyanin levels in cherry was analyzed. When the two most stablereference genes in flower tissues CYP2and TEF2were used for normalization, expressions ofCHS and MYB10were highest in petal with pink color, and lower expressions were observed inpistil as expected. However, the highest transcript levels of CHS and MYB10were observed inpistil when the least stable gene GAPDH was employed for normalization.3.The fruit malformation of ‘Tieton’ and ‘Hongdeng’ were most severely in all the7investigated genotypes,‘Sunburst’‘Lapins’‘Satohnishiki’ followed,‘Regina’ and ‘Summit’slightly generated.The two most severely were early maturity varieties,while the maturity time ofothers were a little later. To a certain extent, it showed that there were some relationshipsbetween malformation and maturity.4. The primers in the conserved domains were designed through comparing the MADS-boxgene sequences of other plants.1gene fragment with suitable size had be gotten by RT-PCR.Thesimilarity between this fragment and PaMADS3acquired from ‘Lapins’ was99%. It was inferedas part of class E MADS-box gene.5. In order to find the specific expression gene in pistil, the qRT-PCR results of PaMADS2、PaMADS3、PaMADS4and PaMADS5in different tissues of ‘Tieton’ and ‘Sunburst’ wasanalyzed. The relative expression of PaMADS2in petals, stamen and total flower was higher. Itsuggested that PaMADS2belong to class B MADS-box gene. The expression pattern ofPaMADS2showed no tissues specificity. PaMADS3showed highest expression levels in petals,which revealed petal specificity.The expression pattern in two cultivars was similar. The highestexpression of PaMADS4was tested in total flower, and the rest showed lower and closerexpression levels, which showed expression characteristics of class E gene participating inregulating all flower organs formation. PaMADS5showed highest expression levels in pistils andstamen,which revealed pistil and stamen specificity. 6. In order to explore the relationship between expression levels of PaMADS2、PaMADS3、PaMADS4and PaMADS5and malformation generation of cherry fruits, the expression of allfour genes in different flower development stages of ‘Tieton’ and ‘Sunburst’ was ananlyzed.Theexpression modal of PaMADS3/4/5was similar between the two genotypes with different rate onmalformed fruits. That is, PaMADS3with highest expression in petals and lowest expression inpistils; PaMADS4expressed in all tissues but with lowest expression in pistils and PaMADS5with highest expression in pistils especially during full-blooming dates. But the expression ofPaMADS2in the two genotypes. Which showed highest expression in petals of ‘Tieton’ duringfirst flowering, then the expression reduced gradually; but it showed lowest expression in petalsof ‘Sunburst’ during first flowering, then increased the expression gradually. Overall, theexpression law of the four tested genes were similar in pistils related to generation of abnormalfruits from two genotypes which showed significantly different rates of deformed fruits. Itillustrated that the above genes were not key genes regulating pistil and malformed fruitsgeneration. |
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