The Biological Characteristics Of Tomato Yellow Fruited Mutant Yft1 And The Cloning Of The Candidate Gene

Tomato(Solanum lycopersicum)is the second most highly produced vegetable crop worldwide after potato(S.tuberosum),with a production of 164 million tons worldwide.And China is the largest producer with50.5 million tons(http://www.fao.org/corp/statistics/en/).Tomato quality improvement has widely drawn attention from tomato breeders and consumers.Fruit color is one of the most important quality characteristics,which is closely related to fruit maturation,flavor,nutrition and market demand.Tomato fruit coloration depends on the composition of carotenoids,flavonoids and chlorophyll.Among these pigments,the carotenoids forming flower color and fruit color are greatly vital to tomato pollination,seed dispersal and protection from ultraviolet.Meanwhile,the antioxidant activities of the carotenoids can prevent cancer and cardiocerebrovascular disease in humans.To date,the structure genes in carotenoids biosynthesis pathway have been clearly studied.However,the molecular mechanisms of regulating carotenoids biosynthesis and accumulation remain largely unknown.Therefore,elucidating the molecular mechanisms of regulating carotenoids biosynthesis and accumulation can provide theory evidence for targeting improvement of tomato quality.In this thesis,we employed a novel tomato mutant n3122 named as yellow fruited tomato 1(yft1)which was derived from the fast-neutron irradiation of the wild type cultivated tomato variety M82.We investigated the characteristics of yft1 from the perspectives of morphology,biochemistry,structural biology and molecular level.We also determined the genetic inheritance of the yellow fruit color phenotype in yft1,as well as map-based cloned the yft1 locus mainly with CAPS molecular markers(cleaved amplified polymorphic sequences).The results were concluded as follows:1 The fruit development of yft1 was delayed.Through observation of fruit development,the time required to reach MG(mature Green),BR(breaker)and RR(red ripening)stages was respectively 35 DPA(day post anthesis),47 DPA and 54 DPA for M82.While for yft1,although it reached MG stage at 35 DPA,it did not reach BR stage until 60 DPA.And yft1 fruits finally displayed yellow color and remained much stiffer.2 The carotenoid accumulation was inhibited in yft1 fruits.Carotenoid was analyzed in different developmental stages of M82 and yft1 fruits by HPLC(high performance liquid chromatography).In M82fruits,the total carotenoids were accumulated with fruit development from 5.07?g·g^-1 FW at 35 DPA to 102.76?g·g^-1 FW at 54 DPA,and lycopene which was undetectable at 35 DPA increased to 85.62?g·g^-1FW at 54 DPA.While carotenoids maintained a steady low level of about5?7?g·g^-1 FW in yft1 fruits during all developmental stages and lycopene was totally devoid.3 The developmental transition of chloroplasts into chromoplasts was hindered in yft1 fruits.Plastid structure was investigated in different developmental stages of M82 and yft1 fruits by TEM(transmission electron microscope).The results showed that plastids in 47 DPA fruits of M82 exhibited a state of chloroplast-to-chromoplast transition and in 50 DPA fruits of M82 grana and stroma lamella representing the typical chloroplast disappeared completely,while carotenoids crystalloids and undulating membrane indicating crystalloids once existed clearly appeared.However,plastids transition proceeded very slowly in yft1 fruits and until 54 DPA that grana and stroma lamella disappeared and no crystalloids appeared all the time.4 Ethylene synthesis and ethylene sensitivity were impaired.Ethylene evolution was measured in different developmental stages of M82 and yft1 fruits by GC(gas chromatography).The results indicated that ethylene evolution increased sharply and peaked at 47 DPA in M82fruits(7.33 n L·g^-1·h^-1),while yft1 displayed a slow increasing pattern and until 60 DPA that ethylene evolution blusted and peaked(5.20 n L·g^-1·h^-1).The peak of yft1 was about 70.1 percent of the peak of M82.To assess ethylene perception,MG fruits were exposed to exogenous ethylene.The results showed that exogenous ethylene accelerated the fruit color change of both M82 and yft1 fruits.However,M82 fruits reached the BR stage on day 7 after treatment.The treated yft1 fruits took13 days to transition from MG to BR.To test the sensitivity of the seedling to ethylene,an ethylene triple response assay was conducted.Roots and hypocotyls of seedlings in both genotypes grown with ACC were significantly shorter than those grown without ACC(p<0.001).But hypocotyls and roots of yft1 seedlings grown with ACC were significantly longer than those of M82 grown under the same condition(p<0.001).5 A single recessive nuclear gene determined the yellow fruit phenotype of yft1.An intra-specific cross(M82×yft1)and an inter-specific cross(yft1×LA1585)were constructed.All fruits from both crosses appeared red(in the F₁ generation,whereas both red and yellow fruit were produced by plants of the F₂ generation,and the chi-square(?²)values of red fruited plants:yellow fruited plants fit the theoretical Mendelian ratio of 3:1,suggesting that the phenotype is controlled by a single recessive gene.6 EIN2 is the candidate gene for the yellow fruit phenotype of the mutant yft1.The F₂ generation(yft1×LA1585)was used as a mapping population.With map-based clonging method,the mutated gene was finally assigned to an 88.2 kb region between CAPS markers 9-S and 9-R.According to the tomato genome annotation,a total of 12 genes are located in this target region;however,of these,only one,Solyc09g007870,which is annotated as encoding the ETHYLENE INSNSITIVE2(EIN2)protein,has been shown to be essential for the ethylene signal transduction pathway and to be involved in regulating fruit ripening.Nucleotide sequencing revealed that a 13 bp deletion and a 573 bp insertion were found at-318 bp upstream of the translation initiation codon of EIN2 in yft1.Moreover,EIN2 expression was significantly down-regulated in yft1at all tested time points between 35 DPA and 60 DPA.7 EIN2 widely affects tomato fruit ripening via regulation ethylene signal transduction.Transcriptome sequencing of different developmental stages of M82 and yft1 fruits was performed by Illumina HiSeq 2000.After processing and analyzing crude data with bioinformatic methods,31 M reads for each sample were produced making up 25548 unigenes totally.5568DEGs(differentially expressed gene)were screened out in the four pairwise comparisons of M82-35DPA/yft1-35DPA,M82-47DPA/yft1-47DPA,M82-54DPA/yft1-54DPA and M82-47DPA/yft1-60DPA.The DGEs were analyzed by GO term enrichment and KEGG pathway enrichment.The results manifested M82 and yft1 displayed great difference at 47 DPA and 54 DPA,since the numbers of DEGs were largest in these two stages,and genes related to carotenoids biosynthesis,ethylene biosynthesis,plastid structure and fruit ripening were screened out as DEGs as well as relevant GO terms and KEGG pathways were enriched.This result coincided with the expremental results of carotenoid content,ethylene evolution,and plastid structure,which implied that EIN2 widely affects tomato fruit ripening process including carotenoids biosynthesis via regulating ethylene signal transduction.This research provided important information for further elucidating the molecular mechanism of tomato fruit color formation including the regulational network of carotenoids biosynthesis and accumulation,as well as precisely improving tomato quality.Meanwhile,it provided important theory reference for elucidating the molecular mechanism of coloration of other berry fruits.