Clone,Expression Pattern Analysis Of WRKY6 Gene Of Tobacco

Plants have been exerted effects during its growth and development by biotic and abiotic stress. To adjust these stresses, plants have developed a series of complicated, systematic regulation system. Among them, WRKY family plays a important role, which includes regulation of expression of stress related genes cope with the environment. With many different bio-functions, WRKY is a plant-specific transcription factor, which is critical for plant stress response, growth, development and morphogenesis. The research of WRKY has made a great progress in Arabidopsis, rice, soybean and wheat already while there’s little report about WRKY of tobacco. In this study, I have cloned a WRKY family member, and performed functional analysis with bioinformatics and expression pattern analysis. The results are as follow:(1) NtWRKY6 was cloned through homology cloning from NaWRKY6 of Nicotiana attenuate. Based on bioinformatics results, the coding sequence of NtWRKY6 was 1647 bp, encoded 548 amino acids and contained 2 WRKY domain, which belonged to class I WRKY protein. Alignment results showed that the gene has more than 87% similarity with WRKY in pepper, tomato, and 97% with WRKY with NtWRKY1. Since there’s high similarity of both AtWRKY6 and NaWRKY6, thus the gene was named as NtWRKY6.(2) The NtWRKY6 was transformed into vector pGBKT7, which further transformed into yeast line AH 109. The transcription activity was further proved.(3) Semi-quantity PCR was performed for analysis of the response of NtWRKY6 on abiotic stress. And it turned out that with potassium deficient stress, the expression level of NtWRKY6 reached its peak within 3h, started to decrease after 3h, and keep stable after 12h. This indicated that NtWRKY6 could response to potassium deficient stress; under phosphorus deficient stress, the expression of NtWRKY6 increased after 12h treatment and reached to its peak at 24h, indicating the inducible expression of NtWRKY6; with sanility stress, the expression of NtWRKY6 started to increase after 3h, and reach to stable stage after 12h, indicating that NtWRKY6 was induced by salinity stress; under drought stress with 10%PEG treatment, the difference of expression of NtWRKY6 was not significant.(4) Over-expression vector for NtWRKY6 was constructed and transformed into Agrobacterium LBA4404. The transformation results were checked by colony-PCR. Transgenic tobacco was transformed through leaf-disc method and screened with Kan. The preliminary results showed that compared with wild type, the expression level of stress-resistant genes in transgenic tobacco was higher. These transgenic tobaccos laid a solid foundation for further functional characterization.