Cloning And Function Study Of Key Genes For Sucrose Accumulation In Pear Fruits

Sucrose is a soluble sugar whose content varies greatly in different varieties of pear fruits,and it is also an important part of the flavor of pear fruits.Therefore,analyzing the molecular mechanism of sucrose accumulation in pear fruit can provide an important theoretical basis for improving the quality of pear fruit.In this study,sucrose phosphate synthase gene(Ppy SPS3 and Ppy SPS8),fructose kinase gene(Ppy FRK5)and related transcription factors(Ppyb ZIP43 and Ppy STOP1)involved in sucrose accumulation in pear fruits were cloned from‘Hosui’pear,and their biological functions in sucrose accumulation in pear fruits were studied.The main results are as follows:1.In previous studies,seven key candidate genes were identified that were closely related to the sucrose content of pear fruits,namely FRK,SUS2,SPS3,SPS8,b ZIP,C2H2 and ERF.In this study,by analyzing the relationship between the transcription levels of these 7 genes and the sucrose content at different developmental stages in 6 pear varieties,it was found that the transcription levels of almost all genes were significantly related to the sucrose content across varieties.Tissue specific expression analysis showed that the transcription level of Ppy SPS3 and Ppy SPS8 was low in the early stage of fruit development of ‘Hosui’ pear fruit,but the transcription level increased sharply in the later stage of fruit development.Correlation analysis showed that the transcription levels of Ppy SPS3 and Ppy SPS8 were significantly positively correlated with sucrose content during the development of the ‘Hosui’ pear fruit.Amino acid sequence analysis showed that both contain typical conserved domains of the SPS family.At the same time,using the techniques of transient overexpression and silent expression in pear fruit,it is found that Ppy SPS3 and Ppy SPS8 can affect the sucrose content in pear fruit by changing the activity of SPS enzyme,thus confirming that Ppy SPS3 and Ppy SPS8 play an important role in the accumulation of sucrose in pear fruit.2.Fructokinase can specifically phosphorylate fructose to produce Fructose-6-phosphate(F6P),and F6 P is one of the main substrates for sucrose synthesis.During the fruit development of ‘Hosui’ pear,it was found that there was a significant positive correlation between the transcription level of Ppy FRK5 and the sucrose content.Amino acid sequence analysis showed that Ppy FRK5 belongs to the pf KB family,has a highly conserved characteristic domain of the pf KB family,and also contains FRK-specific ATP binding domain and sugar binding domain.Subcellular localization showed that Ppy FRK5 was localized in the cytoplasm and cell membrane.The transient overexpression of Ppy FRK5 in the ‘Hosui’ pear fruit can significantly increase the sucrose content in the pear fruit;while the sucrose content in the pear fruit that silently expresses Ppy FRK5 is significantly reduced.These results indicate that Ppy FRK5 participates in and plays an important role in the accumulation of sucrose in pear fruits.3.In order to study the regulatory effects of Ppyb ZIP43 and Ppy STOP1 in the accumulation of sucrose in pear fruits,they were cloned and expressed respectively.Amino acid sequence analysis showed that Ppyb ZIP43 contains a typical b ZIP conserved domain,which belongs to the plant G-box binding transcription factor;Ppy STOP1 contains three zf-C2H2 conserved domains,which belongs to the C2H2 zinc finger protein transcription factor.The subcellular location showed that both Ppyb ZIP43 and Ppy STOP1 were located in the nucleus.Transient overexpression of Ppyb ZIP43 and Ppy STOP1 in pear fruits,respectively,can significantly increase the sucrose content in pear fruits.In addition,transient overexpression of Ppyb ZIP43 also significantly increased the expression of Ppy SPS3 and Ppy SPS8.Yeast one-hybrid and DLR assay results confirmed that Ppyb ZIP43 can bind to the promoter of Ppy SPS3 to regulate its expression.Yeast two-hybrid,BIFC,LCI assay and Pull-down results show that there is an interaction between Ppyb ZIP43 and Ppy STOP1.The above results indicate that Ppyb ZIP43,Ppy STOP1 and Ppy SPS3 form a regulatory module to participate in the accumulation of sucrose in pear fruits.