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Construction of a cDNA library of Bemisia tabac i for use in the ‘yeast two‐hybrid screen’ method
Author(s) -
Ohnesorge S.,
Bejarano E. R.
Publication year - 2002
Publication title -
eppo bulletin
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.327
H-Index - 36
eISSN - 1365-2338
pISSN - 0250-8052
DOI - 10.1046/j.1365-2338.2002.00556_4.x
Subject(s) - insert (composites) , plasmid , biology , cdna library , complementary dna , vector (molecular biology) , whitefly , two hybrid screening , yeast , gene , virology , genetics , recombinant dna , botany , mechanical engineering , engineering
The molecular mechanisms involved in the circulative, non‐propagative transmission pathway of TYLCV through its vector the whitefly Bemisia tabaci have hardly been studied. Points requiring investigation include the specific adhesion of virus coat protein to insect structures, the proteins involved in membrane passage in the insect and the possibility of replication of the virus in the vector. To isolate the insect proteins which are involved in transmission by interaction with viral proteins, we propose to use the ‘yeast two‐hybrid screen’ genetic method. For this method, it is indispensable to have a ‘cDNA library’ of the organism concerned, cloned in plasmids, and our first step has been to develop this. A new method was developed for isolating whitefly mRNA. From this mRNA, cDNA was synthesized, ligated in the plasmid pGADT7 (Clontech) and transformed in bacteria to amplify the plasmid DNA. The number of independent clones and average insert size of the plasmids were determined.