Establishment Of Virus-induced Gene Silencing(VIGS) In Gerbera Jamesonii ‘Hualong' And Its Application In Gene Functional Analysis

Gerbera?Gerbera jamesonii Bolus?is one of the five fresh cut flowers,and it was cultivated widely around the world.While,there were few Gerbera cultivars with our own intellectual property rights and popular in Chinese markets.Traditional breeding methods with mutation,radiation or cross breeding,which have disadvantages with long cycling and lacking of direction.Molecular breeding is not only can greatly shorten the breeding period,but also can be directed to improve the important ornamental traits.And molecular breeding needs to identify the function of genes,however,Gerbera jamsonii as the non-model species,there is a big challenge for it to research the genes'function.Virus-induced Gene Silencing?VIGS?with its merits,which are short period,high throughout,no need for genetic transformation,et al.,were widely applied to identify the functions of genes.In this study,we applied high performance liquid chromatography?HPLC?to detect the contents of anthyocyanins in Gerbera jamsonii Taiwan 509 and new cultivar of its color mutation,named gerbera‘Hualong'.And applied qRT-PCR to verify the differentially expressed genes,which were identified early in our laboratory.Selected 6genes among these differentially expressed genes,to research their expression pattern in process of flower buds'development.Then we established the VIGS system of gerbera‘Hualong',and applied it to analysis the potential functions of these differentially expressed genes.The main result we obtained were as follows.1?Applied the high performance liquid chromatography?HPLC?to detect the contents of five kinds of anthocyanins in Gerbera jamesonii‘Hualong'and Taiwan 509.The result showed that,Pelargonidin 3,5-glucoside chloride?Pel 3,5-glu?,Petunidin 3-glucoside chloride?Pet 3-glu?,Cyanidin 3-glucoside chloride?Cya 3-glu?,Maldivin?Mal?,these four anthocyanins were contained in both two gerbera cultivars.And the contents of these four detected anthocyanins were showed higher in gerbera‘Hualong'than gerbera‘Taiwan509'.The content of Cyanidin 3-glucoside chloride in two gerbera cultivars were both higher than the contents of other 3 anthocyanins?Pelargonidin 3,5-glucoside chloride,Petunidin3-glucoside chloride,Maldivin?,high about 10 times,20 times and 3 times,respectively.But Peonidin?Peo?was not detected in these two Gerbera jamsonii cultivars.2?Eleven differentially expressed genes were selected for the quantitative real-time PCR analysis using the same RNA sample as for deep sequencing,including CHS genes?3members,2 pieces of CHS1 and CHS4?,and one of each for F3H,F3'H,F3'5'H,ANS,UFGT,ANT,PDS,and GST genes.The results comfired the results obtained from deep sequencing analysis and showed similar trends in up or down regulated unigenes:F3'5'H gene was down-regulated expression,other 10 genes were up-regulated expression.Genes expression in different stage were related to the formation of the flowers'colors.The expression of accumulation of GhCHS1 and GhCHS4 were low in front stage?1-4 stage?,then increased gradually;the top expression of GhCHS1 was in the 7^th stage of flowers'development,and the top expression of GhCHS4 wea in the 10^th stage of flowers'development,then they showed a downward trend.The exprission of GhF3H,GhGST,GhF3'H,GhUFGT almost expressed through the whole stages of flowers'development.GhF3H,GhGST,GhUFGT,the top expression of these three genes were in the 10^th stage of flowers'development.And the expression pattern of GhF3H,GhGST,GhUFGT showed up-regulated expression then down-regulated expression,which related to the aging process in 11^th stage of flowers'development.The expression pattern of GhF3'H was out of relative regularity,its expression of every stage of flowers'development had high relative expression,which might have higher gene transcriptional activity.3?Had established TRV2-LIC-GhPDS recombinant virus vetor to infect the young plant leaves of gerbera‘Hualong'.After silenced Gh PDS,the leaves showed photobleaching phenomenon,which means“VIGS”system was established successfully.Then applied Virus-induced gene silencing?VIGS?technology to silence GhCHS1?GhCHS4?GhF3H?GhF3'H?GhUFGT?GhGST.After analyzing we found that,the color of ligulate petals of gerbera‘Hualong'showed light color of different level after silencing genes.And the expression level of target genes were lowered to some degree?83%-95%?.CHSs and F3H genes were the key genes,which dominated the accumulation of the biosynthesis of anthocyanin in gerbera‘Hualong'.They promoted the accumulation of flavodoxine effectively.And we suggested that,the formation of the color of orange gerbera's ligulate petals in front stages were mainly controled by CHS1,which promoted the accumulation of flavodoxine.The accumulation of anthocyanin in late stages were mainly control by expression of CHS4.F3'H and UFGT were with high transcriptional activity,which were benefits to catalyzing the matter of biosynthesis of colored Cyanidin and the accumulation of stable expression material of glycosylated anthocyanin.That's colored gerbera‘Hulaong'petals.GST was mainly involved in the anthocyanin's transhipment of Gerbera jamsonii‘Hualong',and its differential expression was one of the main reason of the floral color variation in Gerbera jamsonii‘Hualong'.In this study,we have established the VIGS system of Gerbera jamsonii‘Hualong'and applied it to research and analysis some related genes of flower color successfully.The successful establishment of VIGS system could be as foundation for researching the genes'function of Gerbera jamsonii with a comprehensive study.And it would implement and promote the rapid of directed molecular breeding of Gerbera jamsonii.