Functional Analysis And Construction Of Fruit Specific Expression Vector Of INH In Tomato

Invertase inhibitor (INVINH) is the new proteins encoded by small gene family in higher plants, it play an important role of research on plant photosynthesis functioning sugar organ development and quality of plant products at level of post-translational in recent years. However INVINH genetic variation in a larger process of evolution, it's only the beginning of physiological role in gene cloning and description. Our laboratory has cloned invertase inhibitor INH gene in different parts of tomato at different periods through RT-PCR. To further explore the use of transgenic genes in sucrose metabolism of tomato molecular mechanisms to establish the basis of information. The fruit specific promoter 2A11 was use to control the foreign gene high-effect express in the fruit and the function of the invertase inhibitor gene was identified, the following majorresults:1. The promoter was isolated by PCR from L.Cheesmanii, L.hirsutum, Liaoyuanduoli, Lijiadafen, and Micro-Tom. Primers were designed from the published sequence to amplify two overlapping fragments to from an 868bp promoter region. The fragments were cloned into pBS-T vector and then sequenced. The sequences showed some differences between 151bp and 183bp of the translation start site to the published sequences of 2A11 (DG453963.1) promoter. And homology is not highly from the wild species and cultivated species (about 90%).2. The fruit specific promoter 2A11 fragment was cut by using restriction enzyme BamHâ… and Hindâ…¢and inserted into the vector p1300 to form expression vector p1300-2A11. INH gene and p1300-2A11 were digested by BamHâ… and Kpnâ… , then using T4 DNA ligase recombinant plasmid P1300-2A11-INH, connected the product p1300-2A11-INH transformed into E. coli TOP 10 cells for were detection by PCR and restriction enzyme digestion.3. With the GUS reporter gene of the Agrobacterium as the injected form of the transient transformation for detection, invertase activities were measured. It showed that invertase inhibitor expressed only in fruit, and leaves no expression. But the fruit with un-injected with agrobacterium and injected with empty vector as control, were found that invertase activity was decreased,. Thesethe results show that the invertase inhibitor can depress the activity of invertase.