The Establishment Of The Hairy Root Transformation System Of Codonopsis Pilosula And The Identification Of SPL Family Based On The Whole Genome Of Codonopsis Pilosula And The Functional Study Of CpSPL10

The dry root of Codonopsis pilosula(Franch.)Nannf,C.tangshen Oliv,C.pilosula(Franch.)Nannf.var.modesta(Nannf.)L.T.Shen,belonging to family Campanulaceae,is a well-known traditional Chinese medicine in China(named Dangshen).In recent years,there are more and more reports about Dangshen,but no research on gene function has been reported in these species.SQUAMOSA promoter binding protein-likes(SPLs)are a kind of plant-specific transcription factors(TFs)and play important roles in many processes of growth and development,including promote pollen yield,flower initiation,inhibit sprout regeneration and lateral root growth,regulate secondary metabolites such as anthocyanins and sesquiterpenes etc.The gens encoding SPLs have been identified in many plants including Arabidopsis thaliana,Solanum lycopersicum,Oryza saliva.Capsicum annuum,Vitis vinifera and so on.However,little is known about the SPL genes in C.pilosula.In this study,the hairy root transformation system of C.pilosula was successfully established and stable internal reference genes of C.pilosula were selected.Based on the whole genomic data of C.pilosula(data unpublished),15 SPL genes in C.pilosula were identified and bioinformatics analysis and expression patterns were performed.Four SPLs were cloned and their overexpression vectors were constructed.The function of CpSPL10 was studied.The main results are as follows:1.Agrobacterium rhizogenes strain C58C1 mediated transformation system of C.pilosula hairy root was successfully established.We optimized several steps in the process of C58C1 mediated hairy root induction,including the most appropriate medium,explant type,time for infection and co-cultivation.We achieved an induction rate of up to 100%when the roots of C.pilosula seedlings were used as explants,infected with A.rhizogenes C58C1 harboring pCAMBIA1305 for 5 min,followed by induction on 1/2MS supplemented with 0.2 mg/L naphthylacetic acid and 200 mg/L cefotaxime sodium.The co-transformed hairy roots were confirmed by PCR amplification of hygromycin phosphotransferase Ⅱ gene and histochemical GUS assay,and the efficiency of transformation was 70%and 68.3%,respectively,when no hygromycin selection pressure was exerted.To increase biomass production,we excised and self-propagated the transformed hairy roots,which produced saponins.The biomass of the transformed hairy roots increased as the culture time increased,but the content of the total saponin showed no significant changes.Successful establishment of the in vitro culture system of transgenic hairy root for this species lays the foundation not only for assessing gene expression and function but also for obtaining high levels of secondary metabolites through genetic engineering technology.2.To screen appropriate reference genes in C.pilosula,we applied four different methods--GeNorm,NormFinder,BestKeeper,and RefFinder--to evaluate the stability of 13 candidates:CpEF1Bb,CpCACS,CpF-Box,Cpβ-Tubulin,CpGAPDH,CpActin2,CpAPT1,CpActin7,CpActin8,CpRPL6,CpHAFl,CpTubulin6,and CpUBQ12.Expression was examined by qRT-PCR for various tissue types,chemical treatments,and developmental stages.For all tested samples,CpGAPDH proved to be the most stable.Comprehensive analysis indicated that the most stable internal reference genes were CpF-Box and CpCACS in different tissues and at different developmental stages,respectively.Under NaCl stress,CpAPT1 was the best internal reference gene.For methyl jasmonate and abscisic acid treatments,CpGAPDH and CpF-Box,respectively,presented the highest degree of expression stability.Based on these findings,we chose CpSPL9 as the target gene for validating the suitability of these selected reference genes.All of these results provide a foundation for accurate quantification of expression levels of interested genes in C.pilosula.3.We identified 15 putative SPL genes from the whole genome of C.pilosula,which were supported by confirmation of the SBP domain.Their cDNA length ranges from 477b p to 3267 bp,the length of gDNA is 2322 bp to19029 bp,and the length of amino acid is 159 aa to 1019 aa.The phylogenetic tree of 16 SPLs from Arabidopsis thaliana(AtSPLs)and 15 C.pilosula SPLs(CpSPLs)was constructed by the maximum likelihood algorithm in MEGA X,which demonstrated that 31 SPLs were clustered into 6 groups(G1 to G6).The expression patterns of 15 CpSPLs was investigated in the one-month-old,two-month-old,three-month-old seedlings,and different parts of one-year-old C.pilosula during the flowering period.Besides,CpSPL2,CpSPL6,and CpSPL11 exhibited significant up-regulation under NaCl stress;CpSPL6,CpSPL12,and CpSPL15 exhibited significant up-regulation under ABA treatments;CpSPL6 and CpSPL15 exhibited significant up-regulation under MeJA treatments,while CpSPL5 and CpSPL8 showed significant down-regulation.4.Four CpSPLs(CpSPL2,CpSPL9,CpSPL10,and CpSPL15)were cloned by PCR and and their ORFs were constructed into the overexpression vector pMDC85.The recombinant vector pMDC58-CpSPL10 and empty vector pMDC85 were successfully transformed into A.rhizogenes C58C1,followed by Agrobacterium-mediated transgenic hairy roots induction.7 positive transgenic lines and 3 blank controls were obtained based on Hyg resistance selection and PCR detection.Transgenic lines OE-2,with the highest expression of CpSPL10,was used for phenotypic observation.Compared with the control,the hairy roots overexpressing CpSPL10 grow faster,which indicated that CpSPL10 plays an important role in the growth of root.The results of this study successfully established the hairy root transformation system of C.pilosula,enriched our understanding of the SPL gene family in C.pilosula,and laid the foundation for the future study of C.pilosula at the molecular level.