Isolation And Characterization Of Tobacco Root-specific Promoter

Tobacco is an important and specific economic crop which makes significant contribution to the country’s economic development. However, tobacco often suffers from many diseases and insect pests resulting in a great lose each year. Bacterial wilt disease resulted from Ralstonia solanacearum infection is a vatal factor restricting the development and safety production of the Southern tobacco area, which has not been effectively solved so far. Ralstonia solanacearum is soil-borne disease, primarily invading the roots of tobacco, and it is a key strategy to isolate suitable root-specific promoters that can control exogenous resistance gene expression in root to defend the Ralstonia solanacearum. In this study, we reported that two tobacco root-specific genes and their upstream promoters were cloned and characterized by the method of molecular biology, bioinformatics and genetic engineering. The results are showed as follows.1. Using cDNA microarray analysis, we isolated two differentially expressed genes, named as NtREx1 and NTR6, from tobacco. The cDNA microarray showed that the expression levels of these two genes were expressed specifically very high in root and not in other organs. Moreover, the expressions of NtRExl and NTR6 genes was not influenced by biotic and abiotic stresses placed on leaf. Semi-quantitative RT-PCR were used to identify root-specific expression for NtRExl and NTR6, which also showed a strong expression in root tissue of them, and no expression was detected in leaf, steam, flower and fruit tissues. So NtRExl2 and NTR6 are suitable for upstream promoter isolation.2. The 3’ends and 5’ ends of NtREX1 and NTR6 cDNA fragments were isolated by the methods of 3’RACE and 5’RACE from a tobacco root cDNA library available in our lab, and the full length of cDNA sequences of NtREx1 and NTR6 with 849 bp and 1,565 bp respectively, were derived. It was identified that NtRExl was an extension protein and NTR6 was an unknown protein through NCBI Blastx, and NTR6 had a conserved domain Kelch motif. The full length of these two genes laid the foundation for the further isolation of the upstream promoter sequences.3. Two promoters regions of 1,574bp and 1,735bp of NtRExland NTR6 were isolated by Flinking PCR; Bioinformatic analysis performed showed that NtREXl and NTR6 promoters contained the basic elements:TATA-box, GATA-box, CAAT-box. Movever, noted were some motifs OSE1, OSE2, ROOTMOTIFTAPOX1, SURECOREATSULTR11 which were associated with root expression.4. To analyze the function of the NtRExl and NTR6 promoters, the NtREx1 and NTR6 promoter-driven GUS gene plant expression vectors were constructed and were used to be transformed into tobacco by Agrobacterium tumefaciens. These two plant expression constructs were named PBI121A-P2 and PBI121GUSA-P6.Up to the present, only one report has touched on the root-specific promoter which lacks the practical value. In this study, we cloned two tobacco root-specific promoters; which not only can be applied to the study of tobacco bacterial wilt disease, but can be used to research the root and stem disease. It would have important theoretical and practical values.