Cloning And Functional Analysis Of D27,the Key Gene For Strigolactones Synthesis From Nervilia Fordii

Qingtiankui,one of famous and valuable herbal medicine in Lingnan,comes from Nervilia fordii(Hance)Schltr of Orchidaceae family.Modern chemical and pharmacological studies and clinical application showed that N.fordii has significant effects in curing lung diseases and atypical pneumonia,which led to a large demand of the herb.However,N.fordii has been characterized that the seeds have no endosperm and mainly propagated by asexual reproduction.Each plant only grows one or two tubers per year,and each tuber develops only one leaf.In addition,it has special requirements of growing environment and the wild resources are decreasing with the reduced distributing area.Plant architecture is a biological character directly related to crop yield,and shoot branching is one of the important factors of plant type.Strigolactones(SLs)are newly characterised plant hormones known to be involved in control of branching/tillering.SLs can interact with auxin and abscisic acid on the growth and development of plant,and it can control the initiation of lateral buds.From the perspective of growth and development in plant,NfD27,the key gene for the synthesis of SLs,was cloned from N.fordii,based on the data mining of related genes involved in SLs pathway from the transcriptome data of N.fordii.Using homologous recombination technology,plant expression vectors and protein expression vectors were constructed,transformed to E.coli,tobacco or Arabidopsis deletion mutant;and the subcellular localization of the protein,and in vivo/in vitro activities of NfD27 proteins expressed in prokaryotic systems were explored.The studies in shoot branching of NfD27 laid a foundation for studying the roles of SLs in N.fordii.The current main progresses of this study are as follows:(1)Using RT-PCR and RACE technologies,three NfD27 genes were cloned and named NfD27aL,NfD27aS and NfD27b respectively.The full-length of NfD27aL is 1074 bp,which contains a 765 bp open reading frame(ORF).The full-length of NfD27aS is 834 bp,with a 642 bp ORF.The full-length of NfD27b is 1160 bp,which contains a 774 bp ORF.Bioinformatics analysis of the proteins encoded by NfD27s reveals that NfD27s belong to the DUF4033 superfamily and all are non-secretory proteins,and predicted to locate in chloroplasts.Phylogenetic analysis shows that NfD27aL and NfD27aS were genetically closer to those from dicotyledons such as tomato,tobacco and Arabidopsis thaliana,while NfD27b locates on the same branch with MtD27,that closer to those from monocotyledons such as barley and sugarcane.(2)Using homologous recombination technology,protein expression vectors of pMAL-NfD27aL,pMAL-NfD27aS and pMAL-NfD27b were constructed and transformed into E.coli strain Rossetta(DE3).The proteins MBP-NfD27aL,MBP-NfD27aS and MBP-NfD27b were obtained after induction and purification.All-trans-β-carotene was used as substrates in enzymatic reaction.In vitro analysis showed that all-trans-β-carotene was isomerized to 9-cis-β-carotene and 13-cis-β-carotene by MBP-NfD27s.In addition,protein expression vectors and pAC-BETA were cotransformed into E.coli strain BL21(DE3),and the transfromants of BETA-NfD27aL/BL21(DE3),BETA-NfD27aS/BL21(DE3)and BETA-NfD27b/BL21(DE3)were obtained.The bacteria were collected at 6,18,24,30,42 and 48 h after induction,and the pigments were extracted with acetone.In vivo analysis showed that all-trans-β-carotene was isomerized to 9-cis-β-carotene,13-cis-β-carotene and 15-cis-β-carotene by MBP-NfD27s.The mainly isomeric product of NfD27aL is 9-cis-β-carotene,and the mainly isomeric product of NfD27aS and NJD27b is 13-cis-β-carotene.(3)Plant expression vectors of 35S::NfD27aL-EGFP,35S::NfD27aS-EGFP and 35S::NfD27b-EGFP were constructed by homologous recombination and transformed into EHA105.Subcellular localization observation by transient expression in protoplast of tobacco leaves showed that NfD27s located in chloroplasts and emitted green fluorescence when excited by blue light.The Arabidopsis thaliana d27 mutants were transfected by floral dipping and T0 seeds were collected.Transgenic plants of T1 generation were screened out using MS+Kan medium.Phenotypic observation and functional analysis need further studies.