Research On Genes Related To Fruit Ripening And Softening Of Strawberry

Physiological and molecular mechanism of fruit ripening and softening is one of the hotissues. Generally，fruit softening is the result of the interaction of cell wall degrading enzymes,cell wall modifying enzymes, hydrolase enzyme related to degradation of cell internalsubstances, and these enzymes genes and regulation factors. However, the physiological andbiochemical changes of different species of fruit ripening and softening, and the expressionabundance and period of similar enzymes genes have also large differences. Strawberry(Fragaria ananassa Duch.) is a non climacteric fruit that of ripening and softeningmechanism is different from respiratory climacteric fruit. Moreover, strawberry fruits ofdifferent cultivated varieties lead to different texture, firmness change and softeningproperties in the process of ripening and softening. Therefore, it is necessary that the researchon specific kinds of fruit on the key enzymes or genes regulated by a variety of factors duringdifferent ripening and softening periods. In this paper, the main results were as follows:1. A forward and a reverse suppression subtraction hybridization（SSH）cDNA librarieswith516and524positive clones were constructed respectively using cDNA from strawberry(F. ananassa) large green stage fruit as the tester and cDNA from strawberry turning stagefruit as the driver. After removing repeat and redundancy sequences,707uniESTs including369uniESTs in forward library and338uniESTs in reverse library were obtained. Theassembling provides a total of123contigs and584singletons by cluster analyses of theuniESTs. Nucleotide homology search with Blastn in NCBI non-redundant nucleotidedatabase,537uniESTs (75.95%of total uniESTs) were homologous with known genes.Protein homology search with Blastx in NCBI non-redundant protein database,505uinESTs(71.99%of total uniESTs) were homologous with known proteins. The results of geneontology annotation showed that193uinESTs were involved in biological process with315times，molecular function with332times and cell component with409times respectively.Among these ESTs，putative proteins were related to cell vegetative growth，division，earlyembryonic development，nutrition transport in large green stage library，and related tometabolites biosynthesis，cell wall-related proteins during fruit softening，pigment synthesis，seed maturation in turning stage library. These results were generally consistent with physiological processes during strawberry fruit development and ripening. In two SSHlibraries ten genes including ripening-related protein, PG, MYB, IRL,LEA, ETR1, ARP, WRKY,FaMADS1and FaMADS2genes were analyzed by qRT-PCR in different tissues and stages offruit development and ripening. The information generated in this study provides new clues toaid the understanding of the development and ripening process in F. ananassa fruit.2. Strawberry fruits from two cultivars ’Toyonoka’-softened texture and ’SweetCharlie’-hard texture, which had different texture and storage characteristics, were used asmaterials. The similarities and differences from the two cultivars of change of firmness andpolygalacturonase (PG), pectin lyase (PL), pectin methyl esterase (PME) and cellulase (Cx)activity during fruit development and ripening and softening were studied. The relativeexpression levels of these enzyme genes including PG, PL, PME, β-Gal (β-galactosidase) andEG (endoglucanase) genes were analyzed by qRT-PCR in different tissues and stages of fruitdevelopment and ripening. The results were as follows: fruit firmness decreased rapidly withthe fruit development and ripening.’Toyonoka’ fruit firmness decreased faster than ’SweetCharlie’, when its firmness value is only54.5%of ’Sweet Charlie’ at fruit pink period.Measurement of total PG activity and PG gene expression were performed in two strawberrycultivars with contrasting softening rates. The softer cultivar ’Toyonoka’ fruit PG activityincreased gradually to peak at pink stage with the fruit ripening, and maintained at a higherlevel in postharvest softening period. The change trend of PG gene expression was consistentwith PG enzyme activity. PG activity of the cultivar ’Sweet Charlie’ fruit can be detected atinitial development stage, reach to the peak at white fruit, and decreased gradually during fruitripening, and remained at the lower level during fruit softened completely. To the cultivar’Sweet Charlie’ the change trend of PG gene expression and change trend of PG activity arenot consistent. A method for determination of strawberry fruit PL enzyme activity wasestablished. PL activity was demonstrated in fruits on supplementing the homogenizingmedium with0.02mol·L-1Na-Pi buffer,0.02mol·L-1cysteine-HCl and1%Triton X-100. Theabsorbance value of PL enzyme activity was measured at500nm. Ca2+on PL enzyme activitydetermination of absorbance value had no significant influence. PL enzyme activity was at alower level in the fruit developmental stage, and maintained at a high level in ripening andsoftening stage. The changing trend of PL gene expression had almost the same change trendwith PL enzyme activity, and in contrast to the fruit firmness change trend. This resultshowed strawberry fruit PL enzyme are closely related to fruit ripening and softening.Throughout the ripening and softening process,’Toyonoka’ fruit PME activity was alwayshigher than that of ’Sweet Charlie’. While the PME gene expression pattern of two cultivarwas different,’Toyonoka’ fruit PME gene expression decreased rapidly as the fruit ripening, while the ’Sweet Charlie’ fruit PME gene expression increased rapidly at the green fruit stage,and decreased rapidly followed by the fruit ripening. Cx activity increased progressively withfruit ripening.’Toyonoka’ Cx activity is significantly higher than ’Sweet Charlie’. Inpostharvest process,’Sweet Charlie’ Cx activity decreased rapidly, while ’Toyonoka’ did rise.This showed the role of cellulase may be different from the different texture types of fruit. EGgene relative expression from ’Toyonoka’ is higher than ’Sweet Charlie’ during fruit ripeningand softening.3. The EST sequence of a SSH library constructed as reference sequence, using theRT-PCR and RACE technology cloned the full-length cDNA of PG, PL, β-Gal, EG genesrespectively from pink stage fruit of two strawberry cultivars. Sequence analysis resultsshowed that the two PG gene ORF regions had five single nucleotide mutation locus, lead totwo amino acid mutations. The length of deduced amino acid sequence of ’Toyonoka’ PL wasmore than ’Sweet Charlie’ PL seven amino acids. PL enzyme protein C terminal variationswere larger than other PL protein from different species. A β-gal gene isolated from’Toyonoka’ strawberry fruits was a new member of the β-gal gene families of glycosidehydrolase family35members, including conservative base sequence "GGPIILSQIENEY" anda binding of lactose lectin structural domain. Two β-gal expression patterns tend to be thesame that relative expression rapid rose in fruit ripening process, and reached maximum levelat pink fruit stage and declined red fruit stage. Whether the gene sequence differencesassociated with fruit ripening and softening speed need further analysis.4. Identification of MADS-box transcription factors in Fragaria vesca genome wereperformed using bioinformatics methods to search genomic database．Bioinformatics analysisof MADS-box gene family in F. vesca，including the number of the FvMADS genes, structuretype, sequence characteristic, chromosomal localization and the distribution of conservedmotifs, were carried out for function identification of MADS-box gene in fruit developmentand mature process. Two siRNA plant expression vectors were constructed according to theFaMADS1gene sequence from strawberry fruit, which to lay the foundation for furtherstudies of FaMADS1regulation in strawberry fruit development and ripening.5. The optimal conditions for Agrobacterium-mediated transformation of shrawberry leafexplants were investigated. As a result, the shoot regeneration capacity of cultivar ’Toyonoka’was inhibited on medium containing40mg/L kanamycin and the growth of Agrobactium wasinhibited completely with200mg/L cefotaxine supplemented in the medium. Pre-culture0-2d, infection3to5min, co-culture72h and adding25-100μmol/L acetosyringone contributedto the highest level of transient expression. The strawberry transgenic plants were obtained byscreening with kanamycin resistance selection and detecting with PCR assays. A complete protocol for adventitious shoot regeneration was developed from the leaves of peach rootstock‘Nemaguard’(Prunus persica×P. davidiana) grown in vitro. Non-expanded leaves alongwith their petioles from3-week-old in vitro-grown shoots were used as explants.Regeneration percentage was influenced by plant growth regulators, basal medium, explanttype, dark period, and gelling agents. Optimal regeneration was observed with leaf explantswounded by transverse cuts twice along the midrib and first incubated with abaxial surfacesfacing upward in the dark for3weeks, and then transferred to the light and cultured with theadaxial side in contact with regeneration medium, as seen on1/2MS, woody plant medium orSchenk and Hildebrandt medium supplemented with2.0mg/L thidiazuron,0.1mg/L IBA and0.25%agar. This produced the highest regeneration percentage at71.7%and a mean of5.74±3.24shoots on1/2MS medium. Adventitious shoots were rooted (98.3–100%) and rootedplantlets survived after acclimatization to the greenhouse. In addition, a complete protocol foradventitious shoot regeneration in vitro was developed from the leaves of plum rootstock‘Marianna’(P. cerasifera×P.munsoniana). The first leaves along with their petioles from invitro-grown shoots were used as explants. Percent regeneration was influenced by plantgrowth regulators, basal medium and explant type.The result showed that the regenerationpercentages were higher significantly on1/2MS or WPM medium than on MS medium or SHmedium. Explants of leaves with petiole were more effective than leaves without petiole orpetiole explants in terms of regeneration percentages. This produced the highest regenerationpercentage at81.7%and a mean of7.46±1.38shoots on1/2MS medium supplemented with2.0mg/L thidiazuron,0.1mg/L IBA,0.25%agar and3%sugar. Adventitious shoots wererooted and the better rooting percentage at96.7%were obtained on1/2MS mediumsupplemented with0.5～1.0mg/L IBA. The rooted plantlets survived after acclimatization inthe greenhouse.