The Primary Study Of An Alkaline/neutral Invertase SlCIN3 Gene Function In Tomato

Alkaline/neutral invertase is also named cytoplasmic invertases(CINs).Alkaline/neutral invertase proteins irreversibly cleave sucrose into fructose and glucose,and play important roles in carbohydrate metabolism and plant development.In this study,by bioinformatics analysis,prokaryotic expression,subcellular localization,RNA interference and other methods to explore the function of the alkaline/neutral invertase Sl CIN3 gene in tomato.The main findings are as follows:1)By bioinformatics analyzing of Sl CIN3,we found that it encoded a protein with an isoelectric point of 8.18 and 653 amino acid residues,and it's molecular weight is 74.46 k Da.By constructing a phylogenetic tree,it was found that Sl CIN3 was classified as ? subfamily,and subcellular localization prediction indicated that it was localized in mitochondria.The MEME multiple sequence alignment revealed that the S1CIN3 amino acid sequence contained 12 conserved domains of the GH100 family.There are many hormone-related response elements and stress response-related response elements on the Sl CIN3 promoter sequence.After the treatment of tomato seedlings with Na Cl,PEG and ABA,the expression pattern of Sl CIN3 gene was analyzed,and the expression of Sl CIN3 was induced by high salt,osmotic stress and ABA.2)By analyzing the expression of the alkaline/neutral invertase family genes in tomato fruit ripening period,it was found that the alkaline/neutral invertase gene in tomato was highly expressed in the fruit color changing stage and red ripening stage,among which the expression of the Sl CIN3 was the highest,and its expression in the color-changing stage and the red-maturing stage increased by 4-5 times compared with the green ripening stage.It is speculated that it may be one of the functional genes in the process of fructose accumulation in the late stage of fruit ripening.3)In order to verify the ability of Sl CIN3 in catalyzing the sucrose decomposition,a prokaryotic expression vector was constructed to induce the expression of Sl CIN3 in vitro and obtain purified protein.The results showed that the optimal conditions for the expression of Sl CIN3 were 0.5 mmol?L-1 IPTG,10?,15 h.Sl CIN3 has the ability to cleave sucrose with an optimum enzyme activity p H of 7.5.Sucrose is the only catalytic substrate of Sl CIN3;fructose has a certain inhibitory effect on its activity,and Tris strongly inhibits its activity.4)In this study,Sl CIN3 constructed plant expression vector fused to GFP,was transiently expressed in tobacco mesophyll cells,demonstrated Sl CIN3 protein was localized in mitochondria.5)A total of 61 S1CIN3-RNAi positive plants were obtained by RNA interference and transgenic technology.According to the real-time quantitative analysis of the leaves and fruits of Sl CIN3 silencing transgenic tomato,the expression of Sl CIN3 in leaves and fruits was significantly decreased,and the Sl CIN3 gene was silenced.The effect on the fruit ripening period is small,which mainly reduces the expression of the color change period and the red riping stage.The determination of soluble sugar content in the red ripening stage of the fruit showed that the total sugar content in the silencing of Sl CIN3 strain increased,the sucrose content remained unchanged,and the fructose and glucose content increased significantly.Sl CIN3 negatively regulated the accumulation of hexose during tomato fruit ripening.