Title page for ETD etd-11132008-084612


Type of Document Dissertation
Author Gutierrez Reynoso, Dina Lida
Author's Email Address dgutie1@lsu.edu
URN etd-11132008-084612
Title Molecular Diversity and Coat Protein Expression of Sweet potato leaf curl virus
Degree Doctor of Philosophy (Ph.D.)
Department Plant Pathology & Crop Physiology
Advisory Committee
Advisor Name Title
Rodrigo A. Valverde Committee Chair
Christopher A. Clark Committee Member
Kenneth E. Damann, Jr. Committee Member
Norimoto Murai Committee Member
Joshua D. Detre Dean's Representative
Keywords
  • sweetpotato
  • Ipomoea batatas
  • gene expression
  • geminiviruses
  • coat protein
  • begomoviruses
Date of Defense 2008-11-07
Availability unrestricted
Abstract
Leaf curl virus diseases have been reported in sweetpotato throughout the world. One of the causal agents is Sweet potato leaf curl virus (SPLCV) which belongs to the genus Begomovirus (family Geminiviridae). In the United States, SPLCV has been found infecting an ornamental sweetpotato and several breeding lines but not in sweetpotatoes grown for commercial production. SPLCV does not cause symptoms on Beauregard, the predominant sweetpotato cultivar in the US, but it can reduce its yield. Since SPLCV could become an important constraint for sweetpotato production; diagnosis, identification, and characterization are essential steps to develop an effective management program.

The variability among begomoviruses obtained from 11 sweetpotato genotypes was evaluated through the analysis of the nucleotide sequence of a fragment of the replication-associated protein gene (AC1). Ten of these begomoviruses were closely related to SPLCV and one was closely related to Sweet potato leaf curl Georgia virus (SPLCGV). These results suggest that in the US, SPLCV may be more common in sweetpotato genotypes than SPLCGV. Phylogenetic analysis using the obtained nucleotide sequences of the AC1 and the full length nucleotide sequences of the coat protein gene (AV1) clustered all sweetpotato begomoviruses together. However, SPLCV and SPLCGV were placed in different groups supporting their status as different species.

Serological detection of SPLCV is not currently available due to the difficulties in obtaining purified virions that can be used as antigen for antiserum production. In attempts to obtain the coat protein (CP) of SPLCV for antibody production, primers were designed to amplify the CP gene. This gene was cloned into the expression vector pMAL-c2E, and transformed into E. coli XL1-Blue. After gene induction, a fusion protein of 72 kDa was purified by amylose affinity chromatography. The yield of the purified fusion protein was approximately 200 ėg/liter of bacterial culture. Digestion with enterokinase cleaved the fusion protein into a 42.5 kDa maltose-binding protein and a 29.4 kDa protein. The latter protein was identified by mass spectrometry analysis as the CP of SPLCV.

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