Construction of a cDNA library for the vine mealybug, Planococcus ficus (Signoret)
Date
2008-12
Authors
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Journal ISSN
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Publisher
Stellenbosch : Stellenbosch University
Abstract
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.
Description
Thesis (MSc (Genetics))--Stellenbosch University, 2008.
Keywords
cDNA libraries, Planococcus ficus, Gene libraries, Grapes -- Diseases and pests, RNA, Mealybugs, Dissertations -- Genetics, Theses -- Genetics