Study On The Role Of Strigolactones In Resistance Of Muskmelon To Broomrape And Broomrape Seed Germination

ObjectIn order to clarify role of CmCCD7 and CmCCD8 of muskmelon which is two key genes of strigalactones synthesis pathway and when they were the muskmelon with resistance to Phlipanche aegyptiaca.Meanwhile,for interpret the conditioning and strigolactones roles during P.aegyptiaca seed germination by transcriptome sequencing analysis.Methods:The conserved sequences of CCD7 and CCD8 genes by analysis of other plant species' genes,and the degenerate primers conserved sequences were designed to obtain the conserved sequences.The flanking sequences were amplified through RACE method.Then,the ORF were predicted by ORF finder,nucleic acids information were analysed by DNAMAN,protein physicochemical properties were predictd by Proprarm,secondary and tertiary structures were predicted by using Antheprot and SWISSMODEL,respectively.Homology comparison and phylogenetic analysis was carried out by BLASTN and MEGA.Establishment of the vitro regeneration system for muskmelon variety "huangdanzi" includes determination of the regeneration ability of differently old cotyledon,the effects of different hormones and different hormone concentration on regeneration ability,the effects of Kan and Cef on regeneration ability,screening the optimum elongation and rooting medium.After that,specific fragments of CmCCD7 and CmCCD8 were selected to construct inverted repeat sequences,then the targets sequences were introduced to plant binary vector.Finally,they were transformed into muskmelon by agrobacterium mediated transformation.The obtained RNAi plants were tested by PCR and Southern blotting.IAA and ABA levels were measured by enzyme-linked immunosorbent assay(ELISA).Root exudate of muskmelon was collected,concentrated and purified,then,the 5-deoxystrigol content was determined by UPLC-MS / MS.In addition,the branches and the number of parasitic broomrape were counted for statistical analysis.Dormant,conditioned and GR24 treated Phlipanche seeds were selected for RNA-seq.At first,the acquired data was validated by RT-PCR.Then,the Unigenes was annotated by using NR,Swissprot,GO,COG,KOG and KEGG databases.And the DEGs was enriched by GO and KEGG pathway.Stored substance,nucleic acid,protein synthesis and metabolism-related genes were analyzed.Finally,the hormone related genes were RT-PCR validation and analysis,and the corresponding hormone levels were measured.Results:The full-length cDNA of CmCCD7 and CmCCD8 are 2346 bp and 2153 bp and,containing 1833 bp(KF381339.1)and 1656 bp(KJ473491.1)ORF respectively.The expression level of CmCCD7 and CmCCD8 are highest in root,and lowest in bud,genrally,the relative expression of CmCCD8 is higher than CmCCD7.The molecular weight of CmCCD7 and CmCCD8 encoded protein were 68.60 kD and 61.24 kD respectively.The predicted tertiary structure of CmCCD7 and CmCCD8 protein are highly homolous with RPE65 family.Two-day-old cotyledon has the highest differentiation rate.And the adventitious buds mainly occur near the cotyledon hypocotyl base side.When the concentration of 6-BA is 1.2 mg/l,there is a relatively high induction efficiency.The higher 6-BA/IAA ratio,the more adventitious buds differentiation.And many adventitious roots were produced in hormone-free MS medium.Cef did not affect the adventitious buds induction rate,and 500 mg/l Cef could fully inhibit agrobacterium growth.Kan(100 mg/l)could completely inhibit induction of adventitious buds,so the 100 mg / l will be used as selected concentration.After determined by PCR and Southern blot,13 and 8 transgenic plants were obtained.Each RNAiCmCCD7 and RNAi-CmCCD8 single copy insert plants were selected for the following experiments.The IAA levels among rnai-73,rnai-84 and wild plants were similar.ABA levels in wild and rnai-73 plants were significantly high than in rnai-84 plant.However,we failed to detect 5-deoxystrigol,even in wild muskmelon.Approximate 100 parasites were observed,whichever in wild or RNAi plants.But the number in rnai-84 plant was significantly lower than the other two plants.Totally,there was 19.79 GB Clean data from RNA-seq,Q30 of each sample were more than 88.36%,and 53,921 Unigenes were annotated in nucleic acid databases.There were 694 DEGs between dormant and conditioned seeds,and 4,630 DEGs between conditioned seeds and GR24 treated seeds.Cluster analysis of DEGs among three samples showed 9 Cluster DEGs,maybe they played different roles during different germination stages.And the DEGs among three different stages were enriched in 96 KEGG pathways.During conditioning stage,GA and BR synthesis related genes were activated,and the endogenous hormones content elevated,respectively.From transcriptome annotation data,three strigolactones receptor genes,MAX2 and two KAI2-like genes were scooped.And we found that the germination stimulus receptor-associated genes had been acitived before or at this stage.After GR24 treated,many related GA,ABA,ethylene and BRs biosynthesis or metabolism genes changed significantly,and the trends were consistent with their levels.In different hormone tests,we found fluridone,a carotenoid synthesis inhibitor,could stimulate P.aegyptiaca seeds germination,which may inhibit the biosynthesis of ABA.Conclusion:In this study,two strigolactones biosynthesis genes,CmCCD7 and CmCCD8 were cloned in muskmelon,and the corresponding RNAi plants were obtained.We discovered the effects of two genes on controlling broomrape were not significant.The RNA-seq data of three P.aegyptiaca seeds germination stages showed that the role of conditioning maybe induce GA biosynthesis rather than acitive strigalactone receptor,GR24 perhaps regulate ABA biosynthesis and metabolism to induce P.aegyptiaca seeds germination.