Salvia Universal Stress Protein Gene (smusp1) A Preliminary Study Of The Cloning And Functional

Salvia miltiorrhiza Bunge belonged to Labiatae family has very high ecological adaptability. And it is a widespread herb from tropical to rigid zone,which has universal stress tolerance. Meanwhile,Danshen is an important Chinese medicine plant.Its roots is a well-known traditional Chinese medicine,and wild used for treating coronary heart disease,cerebrovascular disease, hepatitis,heparocirrhosis,chronic renal failure,dysmenorrheal and neuroasthenic insomnia.In recent years,with the development of main chemical compounds and their pharmacological study of Danshen,the exploitation and using of Danshen resources developed fast.And its demands grew bigger and bigger.But nowadays,Danshen's wild resources are insufficient.The cultivars' quality is different and the plant disease and insect pest is heavier year by year.So it is very important to select and cultivate good germplasm with biological technology.The functional genomics study of S. miltiorrhiza was carried out progressively.It is an important way to understand the mechanism of anti-adverse and improve the plant adverse resistance by founding the related new genes.Based on analyzing the EST database we constructed before,a universal stress protein gene was found,its basic gene characterization was understood through gene cloning and expression pattern study.Transgenic and RNAi techniques were used to identify the relationship between universal stress protein gene and the development and adverse resistance of S.miltiorrhiza.This research can provide information to the study of universal stress protein's function in plant.The main results were as follows:1.A unigene was found by screening the EST library we constructed before,which contained a 534 bp-long open reading frame coding for a protein with 177 amino acid residues.This sequence has highly similarity with other USP genes from other species.The conversed domain of universal stress protein A subfamily was found in this protein from the 43th to 165th amino acid residues.So we named this gene SmUSP1.2.The full length sequence of SmUSP1 was acquired from cDNA and DNA by RT-PCR and PCR.This gene is 1537 bp-long and was composed by 5 extrons and 4 introns.There were consensus sequences in the intron border.It was showed that the molecular weight of SmUSP1 was 19.62 kD,the theoretical pI was 5.948.Bioinformatics analysis of SmUSP1 indicated that it was a non-transmembrane,non-signal peptides hydrophilic protein.Alpha helix was the main secondary structure.3.1700 bp 5'-flanking region of SmUSP1 obtained by DNA walking.The cis-acting elements analysis results showed several plant hormones responsive elements,biotic or abiotic responsive elements were found in the promoter region.Based on this,the expression pattern of SmUSP1 gene was analyzed by real-time quantitative PCR.The results indicated that SmUSP1 gene expressed in all S.miltiorrhiza organs but most highly in leaf.Besides,the expression level of SmUSP1 gene was induced by various environmental factors such as dehydration,ABA,SA and mechanical wounding.4.SmUSP1 promoter-gus fusion vector was constructed and transferred into S.miltiorrhiza. The results of gus staining showed that the 5'-flanking region of SmUSP1 had promoter activity.And this activity can be induced by ABA and high temperature.Thus,SmUSP1 promoter belonged to inducible promoter.5.The overexpression and RNAi transgenic plants were obtained by Agrobacterium tumefaciens-meidiated transformation.Unfortunately,there is no morphological trait difference between the transgenic plants and the control.But when they were cultured in 1/2 MS media supplemented with some concentrations of NaCl and mannitol,the root elongation and MDA content showed difference.The overexpression plants grew faster and the MDA content accumulated slower than control.The RNAi transgenic plants were just the reverse.When exposed to air,the water loss rate of overexpression plants was slower than control,while that of RNAi plants was a little quicker. This told us that the SmUSP1 gene might participated in the response reaction of plant stress resistance.