| This experiment was based on the strawberry cultivar‘Bebihoppe’.The gene sequence of Transparent Testa 10（TT10）was obtained by homologous cloning and analyzed for bioinformatic analysis and temporal expression pattern.To elucidate the function of strawberry TT10 gene in the synthesis of proanthocyanidin in strawberry fruits,we silenced and overexpressed the TT10 gene in strawberry fruit by constructing the silence and overexpression vector,and then determined the change of proanthocyanidin monomers content in the fruits.The main results were as below:1、The full coding sequence of strawberry TT10 gene was obtained using homology cloning and the name was FaTT10.The sequence analysis showed that,this gene consisted of a 1704bp CDS,encoded 567 amino acids.The relative molecular weight of the protein encoded by FaTT10 was 62.36kDa,the theory isoelectric point（pI）was 6.69,contained a transmembrane structure.Through comparing the amino acid sequence encoded by FaTT10with other plant lactase amino acid sequence,We found that the strawberry TT10 protein also had 4 typical His-rich Cu2+binding regions of the lactase protein structure.2、The temporal expression pattern of FaTT10 gene was analyzed by real-time fluorescence quantitative PCR.The results showed that,the expression of FaTT10 gene in strawberry tissues was different.The highest expression was found in fruit（small green period）,followed by higher expression in stem,flower,root and stolon,and low expression in leaves.Its expression in strawberry fruits with fruit mature gradually reduced,the highest expression was in small green stage,and the expression was almost zero after the fruit began to turn red.The trend of expression in strawberry fruit was consistent with and trend of proanthocyanidin content in the fruit.3、We successfully constructed the pTRV2-TT10 silent vector and infected the strawberry fruits by injection infiltration.We found that,the average expression of FaTT10gene in the silent group was 59%lower than that in the control group.And the average content of proanthocyanidin monomer in the silent group was 46.7%higher than that in the control group.It was suggested that the silencing FaTT10 gene increased the content of proanthocyanidin monomer.So we speculated that the FaTT10 gene was involved in the polymerization of proanthocyanidin in strawberry fruit.4、We built 35S:TT10 overexpression vector and infected the strawberry fruit.The results showed that,the average expression of FaTT10 gene in the overexpression group was10.8 times that of the control group,and the average content of the proanthocyanidin monomer in the fruit was 22.9%lower than that in the control group.It also proved that the FaTT10 gene was not just involved in the synthesis of proanthocyanidin of strawberry fruit,and its main function was to convert proanthocyanidin monomer into oligomers and polymers by polymerization. |
