Construction of a cDNA library for the vine mealybug, Planococcus ficus (Signoret)

[摘要] The vine mealybug, Planococcus ficus (Signoret), is a severe pest of grapevinein many grape and wine producing countries around the world. It isrenowned not only for the considerable damage it infers to grapevine of itsown accord, but in particular for its role in transmitting deleterious viral diseasessuch as grapevine leafroll disease, Kober stem grooving, Shiraz diseaseand corky bark. Incidentally, it is an exceptionally tenacious antagonist ofgrapevine, being resistant to both chemical and biological control mechanisms.As a result, finding an effective strategy for P. ficus control has become a mainpriority of viticultural industries worldwide.Possible implementation of biotechnological approaches to pest managementhas resulted in a need for P. ficus genetic data - of which there arecurrently very little available. The transcribed genes of an organism can becaptured in a cDNA library, and the sequences of the various transcripts can then be characterized.In this study altogether five cDNA libraries were constructed from thetranscribed sequences of Planococcus ficus (Signoret). Instrumental to theirconstruction was the identification of an RNA extraction protocol that providedlarge quantities of high quality RNA from mealybugs. The five cDNAlibraries were the result of a set of modifications to the Creator™ SMART™cDNA Library Construction Kit (used for Primary Library construction), anddiffered mainly with regards to range of insert sizes they contain. Whereasan abundance of short fragments were found in the Primary Library (42%of screened inserts 60.5 kb, and 20% >1 kb), the Fractionated Libraries containedinserts of specific size ranges that were more-or-less equally represented.The broadest size range was found in Fractionated Library 4, for which a uniformdistribution over the range0.25 kb - 4 kb was observed. Average insertsizes of Fractionated Libraries 1 to 4 were estimated at 0.25 kb, 0.5 kb, 1 kband 2 kb respectively. These results demonstrated the importance of usinga protocol designed to circumvent the bias towards incorporation of shortertranscripts in cDNA libraries.Although the libraries were not exhaustively analyzed, the outcome of apilot investigation indicated that 41% of the submitted sequences had matchesin the non-redundant database of the National Center for Biotechnology Information(NCBI, E-value 6 10-5), and that approximately 82% of these were ofinsect origin. Moreover, two potential targets for an RNAi-mediated approachto P. ficus pest control were identified. With one exception, these sequencesseemed to be unique to arthropods. Future research needs to investigate theefficiency by which these sequences are able to constrain P. ficus proliferation,and their suitability for grapevine transformation.

[发布机构] Stellenbosch University