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

Holm, Kora (2008-12)

Thesis (MSc (Genetics))--Stellenbosch University, 2008.


The vine mealybug, Planococcus ficus (Signoret), is a severe pest of grapevine in many grape and wine producing countries around the world. It is renowned not only for the considerable damage it infers to grapevine of its own accord, but in particular for its role in transmitting deleterious viral diseases such as grapevine leafroll disease, Kober stem grooving, Shiraz disease and corky bark. Incidentally, it is an exceptionally tenacious antagonist of grapevine, being resistant to both chemical and biological control mechanisms. As a result, finding an effective strategy for P. ficus control has become a main priority of viticultural industries worldwide. Possible implementation of biotechnological approaches to pest management has resulted in a need for P. ficus genetic data - of which there are currently very little available. The transcribed genes of an organism can be captured 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 the transcribed sequences of Planococcus ficus (Signoret). Instrumental to their construction was the identification of an RNA extraction protocol that provided large quantities of high quality RNA from mealybugs. The five cDNA libraries were the result of a set of modifications to the Creator™ SMART™ cDNA Library Construction Kit (used for Primary Library construction), and differed mainly with regards to range of insert sizes they contain. Whereas an abundance of short fragments were found in the Primary Library (42% of screened inserts 60.5 kb, and 20% >1 kb), the Fractionated Libraries contained inserts of specific size ranges that were more-or-less equally represented. The broadest size range was found in Fractionated Library 4, for which a uniform distribution over the range 0.25 kb - 4 kb was observed. Average insert sizes of Fractionated Libraries 1 to 4 were estimated at 0.25 kb, 0.5 kb, 1 kb and 2 kb respectively. These results demonstrated the importance of using a protocol designed to circumvent the bias towards incorporation of shorter transcripts in cDNA libraries. Although the libraries were not exhaustively analyzed, the outcome of a pilot investigation indicated that 41% of the submitted sequences had matches in the non-redundant database of the National Center for Biotechnology Information (NCBI, E-value 6 10-5), and that approximately 82% of these were of insect origin. Moreover, two potential targets for an RNAi-mediated approach to P. ficus pest control were identified. With one exception, these sequences seemed to be unique to arthropods. Future research needs to investigate the efficiency by which these sequences are able to constrain P. ficus proliferation, and their suitability for grapevine transformation.

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