Establishment Of VIGS System And Cloning And Functional Analysis Of LhDTX35 Gene In Lilium Spp

Lilium spp.have great variety of colour phenotype,possessing high ornamental value.Anthocyanin is an important component of colour in Lilium.It is synthesized in cytoplasm but should be transported to vacuole to develop colour.MATE(Multidrug and toxic compound extrusion transporters)transporter protein is one of important models of anthocyanin transportation,however it is unclear whether the MATE transporter protein is involved in the anthocyanin transportation in Lilium.In the present study,we cloned the anthocyanin-associative MATE-like candidate full-length cDNA gene screened by RNA-sequence and characterized the gene function.However,it is challenging for Lilium to establish genetic transformation,so it is important to develop tool to characterize the gene function in Lilium.VIGS(virus-induced gene silencing)technique is simple and rapid tool to unravel the gene function.Today,it has not been reported that the TRV-based VIGS was applied to characterize gene function in intact seedling and of colour in Lilium.Therefore,in the present study,it is investigated whether the TRV-based VIGS technique could be used to identify the gene function in Lilium.Additionally,it is established that experiment system of TRV-based VIGS technique is used to characterize gene function of flower colour in Lilium.The work provided technical support for characterization of gene function and played the theory basis on revealing the anthocyanin transport mechanism in Lilium.The main results are as follows:1.Establishment of VIGS system to unravel the gene function of intact seedling in Lilium.we constructed TRV2-LhPDS VIGS vector,the inserted PDS fragment was in conserved domains.Temperature,variety and inoculation method were compared to investigate the effect on the PDS silencing of seedling in Lilium.The appropriate condition for VIGS system of seedling in Lilium is that: ?Raizan 1‘(Lilium×formolongi)was used as the material;the material was grown at 25℃ after inoculation;the inoculation method was used as rubber plus infiltration with needleless syringe.Using the above parameter,TRV2-LhPDS induced the PDS gene silencing in ?Raizan 1‘ and then the photobleaching phenomenon was developed.2.Establishment of VIGS system to unravel the gene function of colour in Lilium.Based on the MYB gene,which is an important transcription factor of flower colour.We constructed the TRV2-MYB VIGS vector.Various inoculation method,the check time of the silencing material were compared to investigate the effect on the VIGS system to unravel the gene function of colour in Lilium.The appropriate condition for VIGS system to unravel the gene function of colour in Lilium were that: the branch with flower buds and pedicels was used as material.When inoculation,the pedicels should be immersed in the Agrobacterium,vacuum-infiltration was maintained for 1 min.The optimal time of testing positive material by PCR was at 7 dpi.3.Gene clone and functional characterization of LhDTX35 responsible for anthocyanin transportation in Lilium.MATE-like LhDTX35 gene associated with transportation was cloned by RACE,which contained 1524 bp open reading frame(ORF)that encoded 507 amino acids.Phylogenetic analysis indicated that LhDTX35 was closely related to AtDTX35.Bioinformatics analysis showed that the LhDTX35 gene encode the hydrophobic membrane protein with 12 putative transmembrane segments and conserve domain of MATE protein.Subcellular localization experiment showed that the LhDTX35 gene expressed at the plasmalemma specifically.RT-qPCR spatio-temporal expression analysis showed that the LhDTX35 expression level is positive related to the changes of anthocyanin content.Complementation test of AtDTX35 mutant showed that overexpressing the LhDTX35 gene could increase the anthocyanin content and color rage of transgenic seedling at the stage of cotyledon.VIGS experiment showed that TRV2-LhDTX35 vector could decrease the LhDTX35 expression,so the tepal cannot develop the anthocyanin phenotype.The above results showed that the protein encoded by LhDTX35 gene belong to the MATE gene family and may be involved in the anthocyanin transportation in Lilium.Additionally,RT-qPCR results showed that expression level of LhDTX35 was low in upper colourless tepals at S1 and S2,while its expression level increase significantly in upper colourless tepals at S3 and then decreased in upper colourless tepal at S4.Complementation test of AtDTX35 mutant showed that the transgenic seedling could recovery the phenotype of sterile fruit pods.The results showed that the protein encoded by LhDTX35 may have another function,predicting that it may be involved in the physiological process such as the pod cracking.