|
Candidate:
JiQiang Yao
Degree
of: Doctor of Philosophy
Department: Biological Sciences
Title:
Genetic Transformation of Tobacco with a Beta-Glucosidase Gene to Induce Constitutive Systemic Acquired Resistance Against Tobacco Mosaic Virus
Date:
Wednesday, March 10, 2004, 9:00 am - 11:00 am
2908 Wood
Committee: Dr. Alexander Enyedi, Chair
Dr. Todd Barkman
Dr. Silvia Rossbach
Dr. Hector Quemada
Abstract:
In tobacco plants, inoculation of TMV (tobacco mosaic virus) leads to the accumulation of SA (salicylic acid). Most of SA is then converted into GSA (SA 2-O-ß-D-glucoside) and stored in the vacuole. While there's much evidence that SA can induce systemic acquired resistance in plants, no physiological activity has been reported for GSA. The goal of this project is to convert GSA back into free SA in order to increase the endogenous SA level in plant's cell. Increased SA level will improve plant's resistance to pathogen. The enzyme responsible for converting GSA to SA is ß-glucosidase. A ß-glucosidase gene from the bacteria Butyrivibrio fibrisolvens H17c was cloned and inserted into the binary vector pGA482 under the control of 35S cauliflower virus prompter. This is the construction of pGlu100 plasmid. In another experiment, pGlu200 plasmid was constructed by adding vacuole-targeting sequence to the ß-glucosidase gene. Then both plasmids will be transformed into Agrobacterium tumefaciens strain LBA4404. The transformed A. tumefaciens will be used to infect tobacco (Nicotiana tabacum cv. Xanthi-nc NN genotype) leaves which typically contain ß-GSA in the vacuole. Transgenic plants were obtained
from the infected leaves by tissue culture technique. PCR analysis identified 53% of both pGlu100 and pGlu200 plants contained the bglA gene. RT-PCR test demonstrated that bglA gene expressed in 71% of pGlu100 and 67% of pGlu200 plants. Seeds from T0 generation was harvested and sowed to get second (T1). The free SA and GSA level was analysed both in T0 and T1 generation. The results showed that while SA level increased in the transgenic plants, GSA level decreased compared with the control plants. The resistance of the T1 generation to TMV was detected by measuring the lesion size after TMV inoculation. The necrosis size was reduced by 29% in pGlu100 plants and 36% in pGlu200 plants. No difference of the SA and GSA level as well as the necrosis size was found to exist between pGlu100 and pGlu200 plants. The inheritance of ß-glucosidase gene was studied by southern blot analysis and the result confirmed that the bglA gene passed on to the T1 generation. The effect of ß-glucosidase gene on the endogenous SA level of the plant's cell, its role in plant's induced resistance to TMV and its potential use in engineering plants were discussed.
|
Related Topics
Main
List of Archives:
Dissertation Defenses
Current
Dissertation Defenses
|
