Generation of Transgene Constructs for Improving Grape Quality and Characterization of two Regulatory Genes in Vitis Species
Date of Graduation
Master of Science in Plant Science
grapevine, promoter, resveratrol, transcription factors, stilbene synthase, ethylene response factors
My thesis research consists of two projects. In the first project, a promoter regulating expression of a ripening-induced protein (RIP) was isolated from Vitis aestivalis ‘Norton’, which is referred to as VaRIP. Sequence analysis indicated that VaRIP shares 98% identical nucleotides with a previously characterized promoter of mrip1 from V. vinifera ‘Merlot’. Three types of DNA constructs were generated for further studying the promoter and for potentially enhancing resveratrol content of grape berry in transgenic grapevines. The first construct will be used to express the stilbene synthase (Vvst 1) constitutively in transgenic grapevines; the second constructs will allow us to identify tissue specificity and temporal regulation of the VaRIP by monitoring expression pattern of the reporter protein, -glucuronidase (GUS) or green fluorescent protein (GFP), that is inserted downstream the promoter VaRIP. The third construct is engineered for expressing Vvst1 in berry tissues during ripening phase. In the second project, two genes encoding ethylene response factors (ERF4 and ERF5) were isolated from the genomic DNA of six selected Vitis species. The alignment of the genomic sequences of the two genes with corresponding cDNA sequences that were obtained from V. aestivalis ‘Norton’ suggests that neither ERF4 nor ERF5 possesses introns. The absence of introns in both ERFs led to the hypothesis that two genes are structurally and functionally conserved. ERF4 from V. acerifolia and V. rupestris do not have the five nucleotides AAAGG in the genomic DNA has compared with the other four species. ERF5 can not be isolated from the genomic DNA of V. acerifolia and V. vulpina by polymerase chain reaction (PCR) using VaERF5-specific primers. ERF5 of V. cinerea is different from ERF5 of the other three Vitis species in that it has a single nucleotide deletion.
© Brijesh Karakkat
Karakkat, Brijesh, "Generation of Transgene Constructs for Improving Grape Quality and Characterization of two Regulatory Genes in Vitis Species" (2005). MSU Graduate Theses. 2802.