Strategies for Making Ti-plasmid based Genetic Constructs Using Patatin Gene Promoters for Functional Characterization

Abstract

Potato (SolanumtuberosumL.) is one of the prominent crops capable of nourishing the world’s population. Food and Agriculture Organisation of the United Nations has acknowledged potato as the food for the future in order to fight global poverty and hunger.Tuberization in potato is a complex developmental process influenced by various metabolic, environmental and developmental signals. Tuberization in potato is accompanied by the formation of starch and expression of relatively abundant proteins. Patatins, in potato are major storage proteins encoded by a multigene family comprising of 10-18 copies per haploid genome. The patatin gene family in potato is distinguished in to two classes: Class-I and Class-II based on their sequence morphology and expression patterns. Many events underlying tuberization aspects in potato remains obscure both at the biochemical and molecular level. The importance of tuber-expressed promoters has drawn a lot of attention in terms of both basic and applied research. Keeping in view of the above, in the present study efforts were made to isolate and characterize different members of Class-I and Class-II patatin gene family. The background work carried out in our laboratory yielded a number of Class-I and Class-II patatin clones with partial coding region designated as PTV03, PTV01, PK03 and PTV04 respectively as revealed through partial sequencing. For functional characterization studies of these clones, only the promoter regions were amplified and cloned in to pUC19 plasmid vector and well characterized through restriction analyses and PCR. One of the Class I patatin clone PK03 was subsequently cloned in to the Agrobacterium based Ti-plasmid vector (pBI121) and well characterized through PCR. The resulting recombinant Ti-plasmids will be used to transform potato systems for functional characterization of the patatin promoters. Moreover, further efforts are required in making binary genetic constructs using the remaining patatin promoters.