Isolation And Characterization Of A Salicylic Acid Induced Gstu19 Promoter In Orange

The soluble glutathione transferases (GSTs) are ubiquitous and encoded by a large and diverse gene family in plants, which can be divided into the phi, tau, theta, zeta,lambda,DHAR (dehydroascorbate reductase) and TCHQD (Tetrachloro hydroquinone dehalogenase) on the basis of sequence identity. The tau classe of Glutathione S-transferases (GSTU) is unique to plants and plays an important role in stress tolerance and secondary metabolism. Previous studies shows that GSTs can be induced by many factors and their early expressing genes can be induced by salicylic acid (SA). The purpose of this study is to isolate an inducible promoter for agricultural production and biological research. In this study, we found that the mRNA of CiGSTU19 in Cara navel orange leaves was rapidly increased within 8 hours after salicylic acid treatment. Then we cloned CiGSTU19 promoter using chromosome walking. A variety of inducible components in the promoter was confirmed by using promoter analysis software PLACE. We constructed three promoter-deleted vectors for further study of the promoter, which were authenticated by using a variety of genetic transformation methods. The main results are as follows:1. CiGSTU19 can be induced by salicylic acid.Quantitative PCR experiments showed that the CiGSTU19 RNA level in Cara navel orange leaves rose rapidly within 8 hours after the leaves treated with 5mmol/L SA, and then returned to normal. This result suggests that GSTU 19 can quickly response to salicylic acid stimulus. Therefore, we infer that CiGSTU19 promoter is a salicylic acid inducible promoter.2. CiGSTU19 promoter has several inducible elements. CiGSTU19 promoter was cloned by using chromosome walking. According to bioinformatics analysis of the promoter, we found that there were several possible inducible elements in the CiGSTU19 promoter, such as W-box (combinded by WRKY transcription factors), ACGT elements (by bZIP class of proteins), and ARR1 binding elements and so on.3. A series of CiGSTU19 promoter-deleted vectors designed for further functional verification of the promoter. Two short inserts (400bp,700bp) of pCAMBIA1391 series vectors were transformed into Arabidopsis. GUS staining results showed that the 300bp promoter sequence was able to start transcription, and the results of sequence analysis showed that-300bp-+118bp promoter region contained three GATA elements, which is also important in CaMV 35S promoter. The results indicated that the-300bp fragment of CiGSTU19 promoter was acting as a strong promoter. Both GFP and GUS expressing in protoplast transient transformation experiments indicated that the elements existing in this 700bp promoter fragment was induced by salicylic acid.4. We also tried a variety of transient expression methods, such as Agrobacterium leaf injection, Agrobacterium-mediated vacuum infiltration and protoplast transient transformation. The results of the first two experiments were not satisfactory, in which GUS staining experiments did not have an active result. That may be due to the reason that transformation rates of the first two methods were not high enough and bacteria entered the interspace of plant tissues but not the cells, so that the vectors can not get a suitable enviroment for expressing. By contrast, protoplast transformation was very efficient and time-effective, only 2-3 days needed. So it is really an ideal method for transient transformation.