Molecular Analysis and Effectiveness Assay of AV1 Gene in Transgenic Tobacco for Resistance to Begomovirus

Abstract

<p>Genetic transformation<br />of tobacco plant using AV1 gene was conducted at<br />the previously experiment and generated transgenic tobacco<br />plants positively carrying the selectable marker nptII gene.<br />The objectives of this experiment were to (1) analyze the<br />presence of Begomovirus AV1 gene in T0 generation putative<br />transgenic tobacco plants using PCR technique with specific<br />primers and its correlation with resistance phenotype, (2)<br />analyze the integration and copy number of the transgene in<br />T0 generation putative transgenic tobacco plants and its<br />correlation with resistance response, (3) screen the T0<br />generation putative transgenic tobacco plants with the target<br />virus infection and to detect the presence of the virus in the<br />transgenic plant tissue using universal primers. PCR<br />detection of AV1 gene in tobacco transgenic was conducted<br />by using specific primer for Begomovirus AV1 gene.<br />Meanwhile, Southern Blot analysis was conducted by using<br />the AV1 gene probe. The effectiveness of AV1 gene in<br />tobacco transgenic was tested by inoculation of target virus<br />using whiteflies vector. Result of the experiments showed<br />that there was a positive correlation between the presence<br />of the AV1 transgene in T0 generation putative transgenic<br />tobacco plants and the resistant phenotype. Transgenic<br />plants with a single copy integration of the transgene<br />exhibited more resistant than the multiple copy one. and<br />non transgenic plant. The resistance as a result of AV1 gene<br />expression was indicated with no symptom in T0 generation<br />transgenic tobacco plants and the accumulation of the virus<br />in the transgenic plants tissue. Northern and Western<br />hybridization analysis need to be perfomed for investigating<br />the presence of mRNA or protein accumulation so that the<br />resistance mechanism of the AV1 gene could be explained<br />more detail.</p>