Studies On Lignin Biosynthesis And Gene Expression Of Key Enzymes During Grafting Healing Of Oriental Melon

Oriental melon(Cucumis melo var.makuwa Makino)is an important horticultural crop.It is widely cultivated in China and Southeast Asia and has high economic value.Due to the shallow root system of oriental melon,problems such as continuous cropping obstacle and soil-borne disease often occur in the actual production process,which decrease the yield and quality of oriental melon.At present,grafting is one of the most effective methods to solve the above problems.In the experiment,thin skin melon ’Yinquan No.1’ was used as scion,white seed pumpkin’Shengzhen No.1’was used as rootstock,and double root tip grafting method was adopted.First of all,to have a clear oriental melon /pumpkin grafting body interface healing three key phases: isolation layer(IL),callus(CA)and the vascular bundle(VB);transcriptome and metabonomics analysis,clear the grafting procession of healing body outlet of differentially expressed genes and differences metabolites.Secondly,ultraviolet spectrophotometry,enzyme-linked immunosorbent assay(ELISA)and real-time fluorescence quantitative assay(q RT-PCR)were used to determine the lignin content,lignin biosynthesis enzyme and gene expression levels,to clarify the expression characteristics of key enzyme genes in lignin biosynthesis pathway during grafting healing process.Finally,virus-induced gene silencing technology was used to inhibit lignin biosynthesis by silencing CAD enzyme gene MELO3C005809.2 and MELO3C019548.2,and lignin staining was performed on the cross section of grafts by paraffin sectioning technology to clarify how lignin biosynthesis affected the grafting healing process.The main research result are as follows:1.Transcriptome and metabonomics detection were carried out at the grafts junction,and KEGG enrichment analysis showed that in ILvs CA period,differentially expressed genes were mainly concentrated in phenylalanine metabolism pathway and carbon metabolism pathway,and differentially expressed metabolites were mainly concentrated in phenylalanine metabolism pathway.In CAvs VB stage,phenylalanine metabolic pathway and linoleic acid metabolic pathway were more enriched with differentially expressed genes,while there were relatively few metabolites with low enrichment degree.The phenylanlanine metabolic pathway is one of the main pathways for differentially expressed genes and differential metabolites enrichment suggesting that phenylalanine plays an important role in grafting healing process.Further study found that phenylalanine metabolic pathway of differentially expressed genes and differences metabolites mainly comments in lignin biosynthesis pathway,differentially expressed genes,respectively,the annotation in lignin biosynthesis pathway of seven enzyme,respectively phenylalanine ammonia-lyase(PAL),4-coumaric acid: Co A ligase(4CL),Hydroxylcinnamoyl-Co A shikimate,Hydroxycinnamaldehyde dehydrogenase(HCALDH),Caffeic acid O-methyltransferase(COMT),Caffeoyl-Co A-O-methyltransferase and Cinnamoyl alcohol dehydrogenase(CAD).A total of five metabolites were annotated in the lignin biosynthesis pathway,namely P-coumaric acid,Caffeic acid,Ferulic acid,P-coumaryol alcohol and Coniferin.2.Through analysis gene of key enzymes in lignin biosynthesis pathway,it was found that PAL,4CL,HCT,COMT and MELO3C014229.2,MELO3C024886.2,MELO3C024861.2,MELO3C025328.2,MELO3C005809.2,MELO3C023272.2 were significantly increased during the formation of isolation layer,indicating that they were involved in regulating the formation of isolation layer.4CL,HCT,COMT,CCOAOMT,MELO3C014222.2,MELO3C022770.2,MELO3C010719.2,MELO3C024861.2,MELO3C034560.2 were significantly increased before callus formation,indicating that they were involved in callus formation.PAL,4CL,CCOAOMT,HCALDH,CAD,MELO3C014229.2,MELO3C004092.2,MELO3C022770.2,MELO3C024886.2,MELO3C014091.2,MELO3C168450.2,MELO3C025328.2,MELO3C005809.2,MELO3C019548.2,MELO3C034560.2,MELO3C023272.2 are displayed before vascular bundle connection,indicating that it is involved in regulating the connection of vascular bundles.The lignin biosynthetase enzymes made the lignin content accumulate at the grafted interface and reach the maximum on 3 day after grafting(isolation layer),the 6 day after grafting(callus)and the 9 day after grafting(vascular bundle),respectively,which promoted the healing of the grafted critical period.3.Using virus-induced gene silencing technique(VIGS)to silence the CAD enzyme gene MELO3C005809.2 and MELO3C019548.2 in the lignin biosynthesis pathway,the lignin content in the stems of oriental melon was significantly reduced,the silent MELO3C005809.2 gene lignin decrease after more than 1 times,after the screening to use silence MELO3C005809.2 gene of oriental melon seedlings and grafting with white pumpkin seed,the stock then to lignin staining to observe the graft.It was found that CK on the 2 day after grafting,scion and rootstock adhered to each other to form an isolation layer,and callus was formed on the 6 day after grafting,and the lignin at the junction was stained deeply,indicating that lignin accumulated at the junction.And body joints after VIGS grafting isolation layer and the callus formation process is slow,grafting anvil ear not adhesion formation 2 day after grafting isolation layer,and a scion site interface lignin staining shallow,that interfaces to lignin content is low,isolation layer in form 3 day after grafting,7 day after grafting callus formation,its isolation layer formation and callus formation than CK delayed 1 day healing process.The connection time of vascular bundle bridge at grafted grafts of CK and VIGS was the same,but the connection degree of vascular bundle bridge after VIGS treatment was lower than that of CK.