Cloning For Anthocyanin Anabolism Genes Of Lycium Ruthenicum Murr And Establishment Of Regeneration System

Lycium ruthenicum Murr is a plant of Lycium L.belongs to Solanaceae.It is frequently adopted by traditional medicines to cure illnesses,such as heart disease,irregular menstruation,menopause and hypertension.The anthocyanin enriched in Lycium ruthenicum Murr's fruits is regarded as the source of the superior pharmacological effects.However,there are fewer researches concerning the genetic mechanism and tissue regeneration of the anthocyanin accumulation.In this article,based on the high-throughput RNA-sequencing and bio-informatic analysis on fruits of Lycium ruthenicum Murr and Lycium chinense Miller,expression and structure differences of anthocyanin metabolism genes in Lycium ruthenicum Murr and Lycium chinense Miller were analyzed.Meanwhile,the target gene was cloned and analyzed.Finally,leaves and stems,two kinds of explants,were applied to set up the regeneration system of the wild Lycium ruthenicum Murr,providing a proper theoretical basis to breed the new variety of Lycium ruthenicum Murr.Experimental results are listed as follows:1?A total number of 192,869 unigenes were assembled in the fruits of Lycium ruthenicum Murr and Lycium chinense Miller through RNA-seq.And 152,209 proteins were predicted by protein databases,like NR,Nt and Swissprot.There were 58,849 genes presenting significantly different gene expressions between Lycium ruthenicum Murr and Lycium chinense Miller,among which 33,070 genes were up-regulated and 25,779 genes were down-regulated in expressions.72,719 genes were annotated in the KEGG pathway,indicating that “flavonoid biosynthesis”,“anthocyanin biosynthesis” and “carotenoid biosynthesis” ranked top positions in the change of metabolic pathway.Transcription factors of anthocyanin anabolism genes,including PAL,C4 H,C4L,CHS,CHI,F3 H,F3'5'H,DFR,LDOX,MYB,and transcription factor of b HLH had 31 homologous genes in the assembly database.However,homologous genes for F3'H and 3GT were not discovered.All the anthocyanin anabolism genes showed a high expression in Lycium ruthenicum Murr,indicating an active biosynthesis of anthocyanin.Moreover,all the transcription factors of MYB showed a remarkably higher expression in Lycium ruthenicum Murr than that in Lycium chinense Miller.MYB is a regulatory gene to adjust the anabolism of the anthocyanin.Transcription of MYB is helpful for anthocyanin anabolism as well as its gene expression and regulation,which may caused by the anthocyanin accumulated in fruits of Lycium ruthenicum Murr.2?Separate Sl AN2,which is a MYB gene to regulate the anthocyanin synthsis of Lycium ruthenicum Murr,according to the RNA-seq results.This gene is 774 bp long,encoding 258 amino acids,with a molecular mass of 29775.84 and isoelectric point of 7.79.Containing 33 negative charge residues and 34 positive charge residues,it is a member of SANT super-gene family.The maximum and minimum value of its protein hydrophobicity is respectively 1.322 and-3.378,with an average hydrophobicity value up to-0.845,indicating that it has a certain hydrophilia.It is speculated that this protein does not have a transmembrane domain and all the actions are performed outside the membrane.According to the molecular evolution analysis,Sl AN2 has a close relationship with MYB genes that can regulate the anabolism of anthocyanin for species of pepper,petunia and eggplant.3?During the course of setting up the regeneration systems of Lycium ruthenicum Murr,experimental curve,MS+2,4-D 2.0mg·L-1 + KT 1mg·L-1 is suitable induced medium the callus induction rate induced by leaves and stems respectively achieved 96% and 94%.In the event of differentiation culture,MS+NAA 2.0 mg·L-1+6-BA 2.0 mg·L-1 is suitable adventitious bud differentiation medium of leave,the differentiation rate up to 94%;MS+NAA 1.5 mg·L-1+6-BA 2.0 mg·L-1 is the suitable adventitious bud differentiation medium of stems,the differentiation rate up to 89%.In the process of rooting culture,MS+NAA 1.0 mg·L-1 the adventitious bud of Lycium ruthenicum Murr reached the maximum root-growing rate,leave-induced and stem-induced adventitious bud rate up to 89% and 86%.The experiment of transplant,The seedlings average survival rate of leaf explants and stems explant is 92% and 88%.