Dentification Of Danshen NOX Family Genes And SmRbohE Cloning

NADPH oxidase(NADPH oxidase,NOX)is one of the sources of reactive oxygen species(ROS),a large number of NOX family members,functional diversity,involved in plant growth,development and stress response signal transduction process,and recent study have shown that NADPH oxidase was related to plant secondary metabolism.In this study,we obtained the complete nucleotide sequences of a SmRbohE gene and cloned it from Salvia miltiorrhiza Bunge cell suspension culture using rapid-amplification of cDNA ends.and the cDNA and amino acid sequence of NOX gene family were analyzed by bioinformatics.We used quantitative reverse-transcription polymerase chain reaction(qRT-PCR)to monitor the expression of the NOX in Salvia miltiorrhiza Bunge cell suspension culture after treatment with salicylic acid(SA),methyl jasmonate(MeJA),hydrogen peroxide(H2O2).And use qRT-PCR to monitor the expression levels of SmRbohA,SmRbohC,SmRbohE in different organs.Construction of SmRbohE overexpression and RNAi(RNAi)vector by leaf disk method,In order to study the function of SmRbohE in the synthesis of salvianolic acid B.The main results were as follows:1.Acording to the specific functional domain of NOX protein,the NOX homologous genes of Salvia miltiorrhiza Bunge were searched on the basis of the existing transcriptome database,and the NOX homologous genes were identified.The SmRbohE gene was supplemented by cDNA rapid amplification technique.In summary,3 Dendrobium NOX homologous genes were identified.2.The bioinformatics predicted the protein domain of SmRbohA,SmRbohC and SmRbohE,and found that all three proteins had the NADPH oxidase domain,and all of them had a transmembrane domain.The SmRbohA has 938 Amino acid,isoelectric point is 9.23,molecular weight is 107303.32,the average hydrophobic is-0.251.Salvia miltiorrhiza RbohC has 935 amino acids,isoelectric point is 9.06,molecular weight is 105232.13,the average hydrophobicity is-0.340.Salvia miltiorrhiza RbohE has 908 amino acids,isoelectric point is 8.98,molecular weight is 101983.07,the average hydrophobic-0.124,Salvia RbohA,RbohC,RbohE is not secreted protein.The similarity of SmRbohA and SiRbohA was 78%,the similarity of SmRbohE and SiRbohE was 85%,and the similarity of SmRbohE and AtRbohE was 64%.The similarity of SmRbohA and SiRbohA was 78%,the similarity of SmRbohA and AtRbohE was 78%.The similarity of SmRbohC and SiRbohC up to 90%,similarity of SmRbohC and AtRbohC up to 68%.3.The SmRbohE gene was cloned and the full length of the cDNA was named SmRbohE.SmRbohE full-length cDNA was 3020 bp,containing a 147bp 5'-URT,146 bp 3-URT and a 2727 bp open reading frame(ORF).4.The expression of SmRboh gene in roots,stems,flowers,leaves and calli of Salvia miltiorrhiza plant was detected by qRT-PCR.The expression of SmRbohA was the highest in leaves and calli,and the expression of SmRbohC was the highest in leaves,and SmRbohE was the highest in leaves and calli.The results showed that salicylic acid,methyl jasmonate and hydrogen peroxide could improve SmRbohA,SmRbohC and SmRbohE significantly at the mRNA level,and the effect of SA and H2O2 was the most significant.5.We constructed over-expression and RNAi vectors of SmRbohE were successfully and transformed to Salvia miltiorrhiza for studing the role of SmRbohE in the synthesis of salvianolic acid B.