The Sequence Analysis And Function Detection Of Xylem Specific Promoter From Poplar

The main purpose of this paper is to detect the xylem-specific promoter in the three sequences of JCesAP,YCesAP and MDCesAP. If the promoters can be used to incorporate Coleoptera-resistance genes and made these genes to be only expressed in the xylem, this will provide scientific basis for controlling the stem-boring pests especially Cerambycidae. Xylem-specific cellulose synthase gene (CesA gene) is confined to developing xylem cells during plant normal growth only and induced in developing phloem fibers undergoing tension stress also. The cis-acting elements of JCesAP, YCesAP and MDCesAP were analyzed by the PLACE database, the plant expression vector pJCesAP-GUS and pYCesAP-GUS were constructed using DNA recombination technology separately, and then all the plant expression vectors including our lab preserving harboring CesAP-GFP/CesAP-GUS were transformed into tobacco using leaf-disk method. The promoter activity and reporter gene expression levels were tested. Selecting Populus tomentosa Stub-D as the experiment materials, CesAP-GFP gene were integrated into Populus tomentosa Stub-D by Agrobacterium-mediated transformation method. Some resistant calli were obtained and observed under fluorescence microscope. The main results were as follows:1. The cis-acting elements of JCesAP, YCesAP and MDCesAP amplified respectively in earlier studies were analyzed by the PLACE database; the results showed that, the three sequences all had the core element of promoter and the elements related to xylem-specific and wound-defense functions.2. The plant expression vector pJCesAP-GUS and pYCesAP-GUS were constructed successfully using GUS as reporter gene. Each gene was promoted with CesA promoter, containing nptâ…¡gene as a selective marker. The plant expression vectors were transformed into Agrobacterium tumefaciens strain AGL-â… .3. Cefotaxime (CTX) critical concentration was 400 mg/L for the tobacco transformation. The plant expressed vector pCesAP-GFP was transformed to Agrobacterium tumefaciens strain AGL-I. GFP expression showed that, the green fluorescence was observed in all transgenic tobacco, it proved all the three sequences were of promoter activity.4. Hygromycin (Hyg) critical concentration was 10 mg/L for the Populus tomentosa Stub-D transformation. pCesAP-GFP gene was transformed to Populus tomentosa Stub-D leaves. Through several transformations and selections, some resistant calli were obtained, green fluorescence was only observed in local tissues of the transgenic calli comparing to negative control and positive control, it proved that the three sequences not only obtain the promoter activities, but also tissue-specific.