Cloning, silencing, and prokaryotic expression of strawberry sucrose synthase gene FaSus1
2017
Hua, L. N. | Zang, M. | Wang, S. F. | Li, Y. Z. | Shen, Y. Y. | Guo, J. X.
The sucrose synthase (Sus) gene is regarded to be involved in strawberry fruit ripening; however, direct molecular and biochemical evidence is lacking. Here, the coding cDNA sequence of FaSus1 was cloned by RT-PCR and both a hairpin-mediated RNA interference pBI121 vector and a recombinant E. coli expression PET28a vector were constructed. These vectors were then used to transform strawberry fruit and for prokaryotic expression in strain BL21, respectively. The results showed that hairpin-mediated RNA silencing of FaSus1 led to a decrease in sucrose content and inhibited fruit ripening. Enzymatic activity analysis of FaSus1 showed that the reaction system contains 0.309 mg of recombinant FaSus1 protein, reaching 0.617 mg/mL. The cleavage activity of the enzyme was 0.25 U with a specific activity of 0.812 U/mg, whilst the synthetic activity of the enzyme was 0.057 U with a specific activity of 0.185 U/mg. This study provides physiological, molecular, and biochemical evidence to demonstrate that the FaSus1 has high sucrolysis activity and plays an important role in the regulation of strawberry fruit ripening.
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