Cloning And Molecular Characterization Of SaLRR Gene In Sedum Alfredii Hance

Sedum alfredii Hance was found to be able to effectively reduce heavy metalpollution and further identified as a plant with Zn/Cd hyper-accumulation traits inChina. It could also serve as potential candidates for phytoremediation and ecologicalrecovery. In this study, we utilized various molecular technologies including real-timequantitative PCR and gene overexpression to uncover the characteristics ofheavy-metal resistance displayed by a leucine-rich protein gene. The full-lengthcDNA and genomic sequence data for the leucine-rich protein gene have beenattained and bioinformatics analysis has been performed. Likewise some preliminaryprotein functions related to heavy-metal resistance have been characterized. Ourfindings would attribute to clarify the Zn/Cd hyper-accumulation mechanisms ofSedum alfredii Hance and provide theoretical guidance for the breeding ofcadmium-resistant transgenic plants.The main findings were listed as follows:1) The gene encoding for the leucine-rich protein (Leucine Rich Repeat, LRR)gene was achieved by PCR amplification and designated as SaLRR. Sequence analysisshowed that full length cDNA was1253bp with an open reading frame of1107bpencoding a protein of368amino acids. Genomic sequence indicated that this genewas not scattered by introns. We also analyzed the amino acid sequence bycorresponding bioinformatics software tools.2) We first treated Sedum alfredii Hance with400μmol·L-1of cadmium chloride(CdCl2), then tissues including root, stem and leaf were sampled and promptly frozenin liquid nitrogen. Total RNAs were extracted from the samples and the quality andquantity were assessed, among which those RNAs with high quality were further usedfor the synthesis of cDNA. With the synthesized cDNA as templates, real-timequantitative PCR was applied to identify the expression profiles of SaLRR. The resultsshowed that in roots and stems, the peak of SaLRR expression level under stress was the time point of12h while that was6h in leaves, implying that this gene may beclassified to be the category of pre-response proteins.3) In order to assess the functions of SaLRR related to cadmium resistance, wefirst constructed the expression vector pYES2.1-SaLRR and then transformed thepositive plasmid into E. coli BL21Star (DE3). The growth curves of clonestransformed with target plasmid under0.8mM CdCl2stress were calculated withclones expressing empty vector as negative control. The results showed that thegrowth rate of E. coli BL21Star (DE3) containing SaLRR genes were higher than thatof controls, indicating that the expression of SaLRR would endow the E. coli withcadmium-resistance to improve the clone survivals.4) SaLRR was further over-expressed in Arabidopsis thaliana by constructing theplant expression vector pCAM2300-SaLRR. The positive transgenic plants werevalidated by RT-PCR to assure that SaLRR had been inserted into the genome ofArabidopsis thaliana. The characterized transgenic and wild-type plants were treatedwith CdCl2and related physiological studies were performed to detect thephysiological and biochemical indexes. The results showed that transgenic plantsexhibited higher cadmium tolerance compared with wild-type Arabidopsis.