Cloning And Functional Characterization Of BjCRP1and Its Promoter From Brassica Juncea L.

The toxiceffects of heavy metal ions on plant’s growth and development are veryobvious, which will change the plant cell structure, cell morphology, inhibit thegrowth of plants, and even lead to the death of plants. Indian mustard canhyperaccumulate and tolerate a range of heavy metal ions, such as Cd, Zn，Pb, Ni, etc.As a model plant for phytoremediation technology, Indian mustard developed anumber of ways to alleviate heavy metal poisoning, such as metal chelation andcompartmentation. BjCRP1is a Cd stress responsive gene cloned from CdCl2stressedIndian mustard by our lab,which belongs to the Ribosomes-associated Membraneprotein4(RAMP4) family. RAMP4is an end anchoring protein, and also it’s thecomponent of endoplasmic reticulum translocation site. RAMP4is composed of66amino acid residues in rat with molecular weight of7KDa. Its homologous genesexist in animals, plants (rice), yeast and worms (c. elegans), and they are also highlyconservative.In this study, the promoter of BjCRP1was cloned from genome DNAsample of Indian mustard, and its cis-acting elements were analyzed bybioinformative software. In the meantime, BjCPR1was transformed into tobacco viaagrobacterium-mediated method.Through gus staining and metal stress experiments,the tissue localization of BjCPR1and its resistance to heavy metals were analized.The results were as follows:1. By using genome walking technology, the promoter of BjCRP1was clonedfrom the genome DNA of Indian mustard. The promoter is733bp long, contains theregulatory element TATA box and CAAT box in the upstream of translation initiationsite (ATG). In addition, it also has the cis-acting elements such as the diseaseresistance, adversity stress, hormone signaling pathways, optical signal pathway,development and heavy metal responding.2. BjCRP1was transformed into tobacco via agrobacterium-mediated method. T0Seeds of BjCRP1plants were obtained through normal cultivation.3. Through the pheotype analysis of BjCRP1tobacco plants and wild type plants,we found that the plant height and leaf area of BjCRP1plants were markedly reduced,while its green leaf number was significantly increased, and the total green leaf areadid not show significant difference. The biomass accumulation of BjCRP1plants wasalso significantly reduced. The results indicate that under normal condition, BjCRP1 overexpression have a negative effect on the plant height and the total biomassaccumulation of transgenic tobacco.4. Under the stress of200mg/L ZnCl2,100μmol/L CdCl2, or200mmol/LCuSO4`5H2O, the BjCRP1plants’ seeds showed higher resistance ability than theseeds of wild type plants, which indicate that the expression of BjCRP1could increaseplants tolerance ability to heavy metal ions.5. Through GUS staining, we detected the expression of BjCRP1in theprocambium of vascular cylinder in root, and its strong expression in the veins of leaf.In flower, BjCRP1expression were detected in anther, stigma and filaments.Thestaining has more in the bottom than that in the upward in filament and petal.