Functional Analysis Of BADH Gene Promoter From Chrysanthemum Lavandulifolium And The Isolation Of Inducible Promoter

Chrysanthemum (Chrysanthemum×morifolium Ramat.) is one of the ten traditional famous flowers in China and the four famous cutting flowers in the world. In recent years, chrysanthemum breeding by genetic transformation has become a hotspot, and some primary achievements have been attained. Studies on transgene expression in chrysanthemum have focused on the CaMV (Cauliflower mosaic virus) 35S promoter, which is a constitutive promoter. It was expression in the various organs and all periods of growth and development in transgenic plants, it would break the metabolic balance and normal growth and development of host plant. Sometimes, it was also cause exogenous gene silence in the transgenic plants. Inducible promoter activity could increase under physical or chemical treatment, and had relatively low expression level at normal conditions, in transgenic plants it caused less ham compared with constitutive promoter. Studies on the use of inducible promoter into chrysanthemum transgenic engineering have important theoretical and practical significance. Betaine aldehyde dehydrogenase (BADH) gene is very important in the anabolism of betaine in many plants, which is induced by drought, salinity, abscisic acid (ABA) and low temperature. In perious work of our group had cloned 4 promoters of BADH gene from Chrysanthemum lavandulifolium, named DBP11, DBP12, DBP21 and DBP22 (GenBank accession number DQ497620-23). The result of transient expression indicated that all the sequences had the function to drive reporter gene. On the base of the previous work, this paper study the expression pattern of the 4 promoters in transgenic plant, and selected the most sensitive promoter to the abiotic stress, to understand the function of cis-acting elements on the promoter. The main results are summarized as follows:1-In this study, we analysis the expression of BADH gene by real-time fluorescence PCR and the content of betaine in leaves from Chrysanthemum lavandulifolium under 400mmol/L NaCl treatment with 0,0.5h,2h,12h, 1d,2d,4d,6d,8d, lOd and 12d, discussed the relationship between BADH gene and betaine. The results showed that, under NaCl treatment both BADH gene expression and the content of betaine had exhibited the tendency that first increased and then decreased. The expression level of BADH gene was down slightly compared with the untreated control at 0.5h and 2h. With the extension of treatment time, the level of BADH gene was continuous increased and reached the maximum at 6d, which were 4.6-higher than untreated control. The activity of BADH gene was decreased gradually after 6d under NaCl treatment. The content of betaine was a sudden increase in NaCl treated with 0.5h, since then the content of betaine fluctuate raise until treatment at 4d reached a maximum. Thereafter the synthesis of betaine was drop drown slowly. The changes between BADH gene and betaine were not at the same time, but had a lag. The BADH gene expression and betaine expression were inhibition by each other. BADH gene expression was significantly increased under 20% PEG-6000 treatment at 0.5h, and then decreased until 1d reached its maximum expression.2.DBP11, DBP12, DBP21 and DBP22 promter were fused with GUSplus reporter gene, to construct of plant expression vectors, the 4 new vectors were then introduced into tobacco. The transgenic plants were treated with NaCl, drought, ABA, SA and low temperature. The results of GUS activities showed that, DBP11, DBP12, DBP21 promoters were responsed to NaCl, drought, ABA and low temperature stresses, and DBP21 promoter was more sensitive than other two promoters. DBP22 promoter was only induced by NaCl and low temperature treatment, and its basic activity was 0.1 times of other three promoters.3. Selected the strongest inducible promoter DBP12, designed different primers according to the cis-acting elements locations on the promoter, using the PCR method to obtain the 5'and 3'end of deletion fragments of DBP12 promoter. New expression vectors were constructed by replacing the CaMV 35S promoter with the 7 deletion fragment sequence respectively to drive the reporter gene GUSplus of the expression vector pCAMBIA1305.2. The new vector was transferred into Agrobacterium to infect leaf disks of Chrysanthemum lavandulifolium. The result of transient expression indicated that the 7 vectors were successfully constructed.4. The 7 vectors were transformed into tobacco, and the transgenic plants were treated with NaCl, drought, ABA and low temperature stresses. The GUS activities illustrated that, there were both inhibitors and enhansers in the sequence of DBP12 promoter. The cis-acting elements, which regulated the BADH gene of Dendranthema lavandulifolium responsed to NaCl, drought, ABA and low temperature stresses were MYB/MYC recognition sites and ACACNNG core. The inhibitors on DBP12 promoter were control the BADH gene responsed to the stress with the cis-acting element at the same time. In addition,5'-UTR had significant function to regulate the BADH gene response to NaCl, drought, ABA and low temperature treatments, and its role of response to low temperature stress was unique.5. Using chromosome walking method isolated 4 DNA fragments in Genome Walker library of Chrysanthemum lavandulifolium, based on 6 EST sequences which were expression under salt stress. The promoter analysis results showed that, the 24.1 (ADP-ribose pyrophosphatase gene) fragment contains cis-acting element that related to water and biotic stress. New expression vector was constructed by replacing the CaMV 35S promoter with the 24.1 fragment sequence to drive the reporter gene GUS of the expression vector pBI121. The new vector was transferred into Agrobacterium to infect leaf disks of Chrysanthemum lavandulifolium and Chrysanthemum. The result of transient expression indicated that the 24.1 sequence had the function to drive reporter gene.This study identified the cis-acting elements that responded to drought, high salt, ABA and low temperature on the promoter sequence of BADH gene from Chrysanthemum lavandulifolium. And the research serves as an important basis for chrysanthemum transgenic engineering.