Studies On The Cellular, Physiological And Molecular Basis Of Large-Fruit Bud Mutant Of ’Nanguoli’

In this thesis, we studied the mechanism of formation of the larged-fruit bud mutation of’Nanguoli’from the cytology, physiology and molecular biology levels. The main results are as follows:1. The results showed that ’Da Nanguoli’ produces more than 2.5-folds larger comparing with the wild-type ’Nanguoli’ in fruit weight and fruit size in maturity, but the stone size between them had not significantly difference in this period, while the hypanthinum thickness showed 1.5-times larger in’Da Nanguoli’. The ploidy of’Da Nanguoli’ has not changed in flow cytometry testing. Cytological observations showed that both of the ’Da Nanguoli’ cell number and cell length was higher than ’Nanguoli’ in different periods of fruit development, with 1.2 and 1.3-times, respectively. These factors were positively related with the fruit weigh. Moreover, there are some differences of cell division between the two cultivars. The sink activity of ’Da Nanguoli’ was significantly higher than which in ’Nanguoli’ in the early stage of fruit development. However, there was no difference in the later stage of fruit development. The net sink in’Da Nanguoli’was significantly higher than which in ’Nanguoli’ in the most of the periods of fruit development. These results showed that the difference in cell number and size in the hypanthinum is the major cause of the difference in fruit size between a large-fruited size spontaneous mutant of ’Da Nanguoli’ and its wild-type cultivar ’Nanguoli’.2. The changes of sugar and acid components in ’Nanguoli’ and ’Da Nanguoli’ during fruit development were studied by HPLC; The contents of sugar, acid, fruit weight, fruit size and fruit shape index were carried correlation analysis. The results showed that changes of components and total contents of sugar and acid were similar in two cultivars at different fruit development stages, the mainly components of the soluble sugar was sorbitol in early stage of fruit development, but glucose and sucrose were rapid accumulated in the late stages of fruit development. Fructose was the mainly component of total sugar at the fruit mature period. Total sugar showed a significant positive correlation with sucrose, glucose, fructose, and a significantly negatively correlated with the shikimic acid, and a negatively correlated with the malic acid and citric acid. The total contents of organic acids in fruits were decreased with the development stages of fruit. The mainly component of organic acids was malic acid in early stage of fruit development, followed by citric acid. Total acid contents showed a significant positive correlation with malic acid content, citric acid content and fruit shape index. Fruit size was positively correlated with the different soluble sugars contents and significantly positively correlated with the fruit, glucose and fructose sugars contents. It indicated that larger size fruit and heavier fruit should be with the higher total sugar contents. Meanwhile, fruit size was negatively correlated with malic acid, shikimic acid and citric acid contents. In addition, fruit weight and fruit size showed a significant negative correlation with fruit shape index, indicated the lower of fruit shape index of the larger of fruit size.3. The cultivars ’Nanguoli’ and ’Da Nanguoli’ were used to investigate the regularity of fruit growth and development, and the changes of endogenous hormones contents during the period of growth and development. The content of endogenous hormones in pulp and seed was determined by using the HPLC. The results showed that the change of content of IAA and ABA in the pulp were almost similar in two cultivars at different fruit development stages. In the first stage of fruit development, IAA and ABA contents were high and then decreased rapidly, and there was no significant difference between IAA and ABA contents in pulp of ’Nanguoli’ and ’Da Nanguoli’ during fruit expanding period. The changes of contents of GA3 and ZT in the pulp were similar in two cultivars at different fruit development stages, and the GA3 and ZT content in pulp of ’Da Nanguoli’ were higher than that ’Nanguoli’. The changes of endogenous hormones contents in the seed were almost similar in the pulp of two cultivars at different fruit development stages; the seed have higher endogenous hormones contents than in pulp. In general, The ratio of IAA/ABA and GA3/ABA in pulp of’ Da Nanguoli’have significantly higher than ’Nanguoli’ at the first stage of fruit development, however, except for between 105 DAF and 115 DAF, there was no significant difference between IAA/ABA and GA3/ABA ratio in pulp of ’Nanguoli’ and ’Da Nanguoli’ during fruit expanding period. Except for 115 DAF, ’Da Nanguoli’ showed consistently higher ratio of ZT/ABA in the pulp than did the ’Nanguoli’ during fruit development. The ratio of IAA/ABA, GA3/ABA and ZT/ABA in seed of ’Da Nanguoli’ showed significantly higher than that of ’Nanguoli’ between 35 DAF and 75 DAF, however, there was no difference between 75 DAF and 135 DAF. In summary, the variation of the fruit development between’Da Nanguoli’ and ’Nanguoli’resulted from the variation of their endogenous hormone contents and balances..4. Based on our results, there were no reliable differences between the’Da Nanguoli’ and ’Nanguoli’on DNA level, by AFLP analysis. The MSAP analysis results showed that the total methylation ratio of ’Nanguoli’and’Da Nanguoli’genomic DNA were 8.98% and 7.80%, respectively, the full methylation and hemi-methylation of two cultivars were 6.67%,5.80%, and 2.31% and 2.00% respectively. Full methylation was higher than hemi-methylation. Compared the level of methylation of’Da Nanguoli’, the hypemetylation ratio of ’Nanguoli’ was 4.16%, the demetylation ratio of ’Nanguoli’ was 1.66%, the methylation ratio that was not changed in two cultivars was 94.18%. These results showed that methylation patterns varied between the two cultivars. Site of the methylation patterns of change may be the cause of’Nanguoli’big sport is one of the causes of occurrence.5. By using cDNA-AFLP combined with qRT-PCR technique, we have detected 98 different TDFs in the expression profiles of fruit development between’Nanguoli’and’Da Nanguoli’, which was considered as the genes regulated the fruit size. Seventy-six of which has been sequenced successfully, the GC contents was 45% among them. Homology comparison analysis shows that 25 TDFs (32.9%) had higher sequence homology protein in the non-redundant protein database, and 31 TDFs (40.8%) had higher sequence homology protein in the non-redundant other EST databases. But other 20 TDFs had not found sequence homology protein and EST. We have also analyzed in the functional annotation, functional classification and metabolism pathway analysis, the result showed that metabolic processes account for almost 55% of the annotated sequences, including primary, macromolecule, cellular metabolic processes and nitrogen compound metabolic process, the biosynthetic processes account for 11.90% and signal transduction account for 3.57%. Furthermore,17 TDFs has been analyzed in qRT-PCR technique, we get the same results with the cDNA-AFLP analysis, indicating the reliability of cDNA-AFLP technique.6. The homology cloning method was used to clone the PNAC gene in’Da Nanguoli’ and’Nanguoli’. Two novel PNAC genes were discovered in’Da Nanguoli’and ’Nanguoli’, and deposited as PNACD and PNACN. The putative physical and chemical properties and functional domains of inferred amino acid sequences of cloned PNACD and PNACN were analyzed, and the alignments and phylogenetic analysis with the other species were also made. The results showed that PNACD and PNACN belong to the NAC family and the gene structure and the characteristic domains in deduced amino acid sequences of the PNACD and PNACN genes were similar to those of NAC genes from other species. They belonged to the NAM subgroup of NAC family. The deduced amino acid sequences of the PNACD and PNACN homology was 84.9%, compared to PNACD, PNACN inserted continually 6 histidines at the 245th amino acid residue.