Cloning And Expression Analysis Of C2-SRC2-like Gene From Salvia Miltiorrhiza

Salvia miltiorrhiza Bunge is the herbal medicines whose dry root and steam is known as traditional Chinese medicine Danshen and Phenolic secondary metabolites are the main medicinal components.Calcium of the plant participate in many signal transduction processes,such as plant growth,development and stress responses,as a second messenger.C2-SRC2-like protein contain C2-SRC2 specific binding sites which play an important role in the calcium ion binding process.We obtained a complete nucleotide sequence of C2-SRC2-like gene and cloned it from Salvia miltiorrhiza Bunge cell suspension culture.And the nucleotide and amino acid sequences were analyzed by bioinformatics software.We used quantitative reverse-transcription polymerase chain reaction(qRT-PCR)to monitor the expression of the C2-SRC2-like after treatment with salicylic acid(SA),calcium chloride(CaCl2)and lack of calcium to Salvia Miltiorrhiza Bunge cell suspension culture.In order to study the function of C2-SRC2-like in the synthesis of phenolic secondary metabolites,the contraction of over-expression and antisense expression vectors of C2-SRC2-like were introduced into S.miltiorrhiza by leaf disk method.The following main findings were obtained:1?The first cDNA encoding C2-SRC2-like was cloned and named C2-SRC2-like gene.The full length of cDNA sequences were 1516 bp with an 1134 bp open reading frame which encodes 377 amino acids,a 109 bp five prime untranslated region and a 273 bp three prime untranslated region.2?Bioinformatics analysis showed that C2-SRC2-like in Salvia miltiorrhiza Bunge had a 38.9 KD molecular weight and 6.9 PI value,which had a transmembrane structure and belonged to hydrophilic protein with no signal peptide.The protein may be located in the nucleus,belonging to the C2 superfamily,and the protein sequence alignment showed 72%similarity to SRC2-like protein of sesame.3?We used qRT-PCR to detect the expression of the C2-SRC2-like after treatment with salicylic acid(SA),calcium chloride(CaCl2)and lack of calcium to Salvia Miltiorrhiza Bunge cell suspension culture.The results showed that the expression of C2-SRC2-like increased significantly with the salicylic acid and excess calcium treatment,and peak appeared at 50 min and 40 min;The expression of C2-SRC2-like continued decreased with the lack of calcium treatment.4?The overexpression and antisense expression vector of C2-SRC2-like gene was successfully constructed,which laid the foundation for the study of C2-SRC2-like protein in the process of phenolic secondary metabolites synthesis.5?The overexpression and antisense expression vector of C2-SRC2-like gene were transferred into Agrobacterium tumefaciens GV3101,and then introduced into Salvia Miltiorrhiza by leaf disk method.The overexpression and antisense expression vectors were transferred into the sterile seedlings of Salvia miltiorrhiza successfully detected at the gene level,prepared for the further study of the function of C2-SRC2-like protein in the accumulation of phenolic secondary metabolites in Salvia Miltiorrhiza.