Enumerating The Expression And Function Of CaPG Gene In The Nightshade Family Solanaceae

International trade in high value perishable members of nightshade family has grownenormously in the past few decades. In the developed world consumers now expect to be ableto eat perishable produce from all parts of the world, and in most cases throughout the year.Perishable plant products are, however, susceptible to physical damage and often have apotential storage life of only a few days. Generally the regular losses caused by post-harvestspoilage of perishable products can be as much as50%. An aspectual understanding, asprovided by this research, is therefore vital in helping to reduce these losses by a significantpercentage. Polygalacturonase a cell wall hydrolytic enzyme is considered one of theparamount agents involved in the deterioration of pectin and cellulose of growing fruits.Initially, the activity and expression of polygalacturonase was determined in capsicumcultivars and then its candidate gene was cloned, manipulated in one of the speciallyengineered expression vector pVBG2307and transferred to the cultivars of capsicum, tomatoand tobacco. Activity and expression of polygalacturonase (PG) a hydrolytic enzyme beinginvolved in ultrastructural changes in the pericarp of sweet pepper (Capsicum annaum L.)were investigated at different ripening stages of two pepper cultivars namely Mandi andTalanduo. Molecular cloning of CaPG from fruits of pepper (Capsicum annaum L. cv. Mandi&cv. Talanduo) was carried out by constructing a cDNA library from three stages of fruitripening. Morphological determination, PG assay, RT-PCR, and ultra-structural studies wereused to quantify changes in CaPG gene expression in pericarp from green, color breaker andfully ripened stages. Results indicated that CaPG gene expression, PG activity and strikingchanges in the structure of the cell wall were occurred with the transition of ripening stages.CaPG gene expression was strongly attributed (obvious PCR products) in mature and ripenedstages of both cultivars whereas, CaPG gene expression was not found in preclimacteric fruitsor vegetative tissues. It was observed that developmental regulation of CaPG gene expressionis instrumental for its role in sweet pepper fruit ripening, and its developmental expressionleads to dissolution of middle lamella and eventually disruption of the fully ripened cell wall. Our studies at molecular and ultra-structural levels have significantly shown that PG exhibitedvariable activity and expression at different stages of sweet pepper fruit ripening. Related tovector pVBG2307molecular chaperones of plasmid pBI121carrying CaMV35S promoterand a nucleotide sequence of plasmid pBI221possessing NOS terminal were inserted intoplasmid pCAMBIA2300to construct an intermediate. We report a novel vector pVBG2307,that contains the significantly expanded multiple cloning site (MCS) with an adjacentimported CaMV35S promoter sequence. This vector allows the controlled transformation ofT-DNA in both Escherichia coli and Agrobacterium tumefaciens. Cloned PG, orf456, iptgenes and E8a special fruit promoter were amplified by conducting PCR on the cDNAlibraries of Capsicum annum and Lycopersicon esculentum then linked into vectorpVBG2307. The usefulness of the vector was demonstrated by regulating PG, orf456, ipt andE8genes in E. coli and transferring into Agrobacterium strain EHA105-4. Agrobacteriummediated transformation of CaPG gene in cultivars of nightshade family B-72, Micro-Tomand Bai Ri-Hong (BRH) was carried out. PCR amplification revealed successfultransformation of aim gene in the cultivar B-72and BRH.