Cloning And Functional Analysis Of SaCAD Gene From Sedum Alfredii Hance

Plant cell walls protect plants from heavy metal stress by resist,tranport and adsorpt process.Cell walls are impregnated with lignin,which enabling structural integrity,long-distance water transport and protect plants from many kinds of stress.Cinnamyl alcohol dehydrogenases(CAD)is an essrntial enzyme paticipated in the final step of phenylpropanoid lignin biosynthesis pathway and plays an important role in anti-stress process.In this paper,we isolated a cadmium stress induced SaCAD gene from hyperaccumulating Sedum alfredii Hance(S.alfredii)combine with relative function analysis.The key results are as follows:1.According to a cadmium resistant related gene fragment from S.alfredii transcriptome database,we amplified the SaCAD full-length c DNA by using 5'RACE and 3'RACE.The 1371 bp cDNA,which with an opening reading frame(ORF)of 1089 bp,encoding 362 amino acids and 38.65 kD of protein.Using PCR amplified genomic DNA sequence,the genome DNA was 2275 bp and containing 5 exons and 4 introns.2.Subcellular localization experiment in Nicotiana benthamiana showed that the SaCAD-GFP fusion protein was mainly located in the cytoplasm.Bioinformatic analysis showed that SaCAD amino acid sequence had the typical conserved domains of alcohol dehydrogenase: Zn1 binding motif,Zn2 binding motif and glycine riched NADPH binding site.Phylogenetic tree analysis showed that SaCAD was closely related to AtCAD7,AtCAD8,CsCAD3 and PtCAD7.3.Real-time quantitative PCR(qRT-PCR)results showed that SaCAD highly expressed in root tissue of S.alfredii.After cadmium(Cd)stress,SaCAD expression in roots was increased first and then declined.The gene expression in stems and leaves were gradually increased with stress time prolonging and dropped after 72 h of stress.It is suggested that the SaCAD is mainly worked in roots and it began to participate in Cd response in stems and leaves when cadmium ions were transported from the roots to the aboveground parts.4.Constructed plant expression vector and transform it into yeast mutant ycf1 and Arabidopsis thaliana(A.thaliana).The results of yeast growth condition showed that SaCAD can not improve the Cd tolerance of yeast.However,we found that transgenic A.thaliana exhibited higher Cd tolerance as compared with wild type(WT),with higher chlorophyll content,proline(Pro)content and antioxidant enzyme activity as well as lower methane dicarboxylic aldehyde(MDA)content,electric conductivity and reactive oxygen species compared to WT when exposed to Cd stress.The Cd cotent determination indicated that the transgenic lines can accumulated a certain amount of Cd.5.We further analyzed the expression of SaCAD and AtCAD gene family members in WT and transgenic Arabidopsis after Cd stress by qRT-PCR.It showed that transgenic Arabidopsis had higher transcript abundance of SaCAD and had more express-advantages under Cd stress compare with AtCADs.Besides,transgenic Arabidopsis had higher CAD enzyme activity with deeper cell wall lignification,and cell wall components like lignin and pectin content were also increased.