| In order to analyze the color changing of yellow-flesh kiwifruit’Jinfeng’in process of fruit development and find out the potential genes related to fruit color controlling in yellow flesh kiwifruit,’Jinfeng’(Actinidia chinensis Planch.), a yellow flesh cultivar, was taken as material to study extraction methods of carotenoid and total RNA from fruit flesh, and conduct correlation analysis for physiological indexes such as chromatism, chlorophyll, carotenoid, carotenoid/chlorophyll (ratio), flavonoid, anthocyanin, sugar, acid and so on by taking green flesh kiwifruit as control. After that, transcriptome sequencing technology was used to sequence the transcriptome of’Jinfeng’and construct Digital Gene Expression7) libraries of three different color changing phases of’Jinfeng’by cooperating with Illimina/Hisseq sequencing technology, so as to analyze the differential expressing genes related to color changing in fruit development, which would provide a theoretic basis for analyzing color changing discipline during fruit development of’Jinfeng’, searching potential and key genes regulating fruit color performing and discussing color performing mechanism of kiwifruit flesh. The results were as follows:1) In this study, microwave method of organic solvent was used to extract carotenoid from Actinidia chinensis Planch, fruits. According to single factor experiments, the results showed that the best single factor for carotenoid extraction was ethanol-acetone mixtures: f=2:1, middle high microwave power, time:20s, ratio of solid to ethanol solution:1:6. Further more, orthogonal test indicated the optimization extraction condition was ratio of solid to ethanol solution:1:6, time:25, middle high microwave power with ethanol-acetone mixtures (f=2:1).2)In order to analyze color changing of ’Jinfeng’ during fruit development, physiological indexes such as chromatism, chlorophyll, carotenoid, carotenoid/chlorophyll (ratio), flavonoid, anthocyanin, sugar and acid was determined by taking ’Jinfeng’ (yellow-flesh) and ’jinkui’(green-flesh) as materials and used to conduct correlation analysis. The results suggested chromatism of ’Jinfeng’ and ’Jinkui’ flesh during fruit development performed significant and negative correlation with ratio of carotenoid/chlorophyll, sugar and acid. Whereas, based on chlorophyll a and chlorophyll b content of’Jinfeng’decreased more rapidly than ’jinkui’ but no differences in carotenoid between mature phase and young fruit phase, we deduced that the decline of chlorophyll and constant content of carotenoid was the important factor for flesh color of’Jinfeng’ being yellow. In addition, according to significant and positive correlation of flavonoid and chromatism, the result that the flavonoid content of’Jinfeng’was higher than ’jinkui’ all the time might be another important factor for’Jinfeng’flesh being yellow. Moreover, flavonoid presented significant and negative correlation with sugar and acid, which suggested that higher content of sugar and acid might exist function, keeping higher flavonoid, so as to affect fruit flesh color. Taken together, the important factors which induce’Jinfeng’flesh being yellow may be due to the degradation of chlorophyll, stable carotenoid, higher flavonoid content, sugar and acid and so on.3) To compare the practicality of total KNA extracting methods for yellow-fleshed kiwifruit fruits that were Guanidine thiocyanate method, Total RNA Extraction Kit and improved CTAB method indicated the improved CTAB method was better than the other two methods. And the A260/A230 was 1.4～1.9, A260/A280 was 1.7～1.9. Further more, the results of Agarose gel electrophoresis and RT-PCR suggested that the total RNA extracted by improved CTAB method could be used for the following molecularbiology operations.4) Taking yellow flesh cultivar ’Jinfeng’ as tested materials, three DGE libraries of different color turning stages constructed by Illimina/Hisseq sequencing technique were used to carry out the enrichment analysis of GO and Pathway function, based on the data of transcriptome. The results showed that transcriptome library analysis generated 47,233 Unigenes, being average length 739bp; blasted with the NR, NT, SwissProt, KEGG, COG and GO databases,34,313,30,203,20,921,18,961,11,660 and 13,905 transcripts were annotated respectively, but 1117 transcripts left unknown. Additionally, combining with the analysis of DGE libraries, there were 4,724,009,4,565,159 and 4,626,730 tags corresponding to the color turning pre-phase, color turning phase and maturing phase respectively, and 2641 genes displayed discrepant expression in color turning pre-phase and color turning phase,2671 genes in color turning pre-phase and mature phase; 1457 genes in color turning phase and mature phase. Moreover, the enrichment analysis of Gene Ontology (GO) and Pathway function appeared that the 12 of the 102 Unigenes on chlorophyll synthesis and 14 of the 111 Unigenes on caroteniod synthesis performed significant discrepancy (P<0.05) in the three color turning phases of ’Jinfeng’ kiwifruit fruit. Subsequently, this study suggested the key genes of fruit color controlling in kiwifruit with yellow flesh might present in the 12 Unigenes related chlorophyll synthesis and 14 Unigenes related caroteniod synthesis. |
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