| Dwarfing is one of the most important traits of plant type.Compact dwarfing of muskmelon plants can increase planting density and economic yield.So far,the molecular mechanism and regulatory pathway of muskmelon dwarfing are not clear.A muskmelon dwarf gene(Cmsi)was obtained by map-based cloning and identification in the early stage of the research group.On this basis,the muskmelon dwarf gene(Cmsi)was analyzed by sequence analysis,spatio-temporal expression analysis and transgenic function verification,and the molecular mechanism of muskmelon dwarf gene(Cmsi)regulating stem development was explored by transcriptome sequencing.The theoretical basis of dwarf plant development of melon was enriched.The main results are as follows:(1)Sequence analysis showed that the full length of CmSI gene was 7128bp,including 27 exons,and the full length of CDS sequence was 2976bp,encoding 991 amino acids.A base substitution mutation(T-G)occurred in the 25th exon of CmSI gene in muskmelon dwarf mutant M406,which led to the early termination of the coding protein.Amino acid sequence alignment analysis showed that CmSI encodes a serine/threonine receptor protein kinase rich in leucine repeat structure of ERECTA-Like,and the mutation of CmSI gene leads to the deletion of protein Kinase kinase domain.(2)The character investigation and data analysis of wild type TopMark and short vine dwarfing material M406 showed that there was no difference in the diameter of main stem and the number of internodes between the two parents,the internode length of the mutant was significantly reduced.Semi-thin section analysis of the parental stem vine found that the Internode cells of the mutant,especially the parenchyma cells,became significantly smaller.(3)The results of qRT-PCR test showed that the expression of CmSI gene decreased significantly in root,stem,leaf,male flower and ovary of short vine dwarfing material M406,and the expression was the highest in stem and ovary of two parents.The results of in situ hybridization and GUS staining also showed that CmSI was abundantly expressed in the epidermis and vascular bundles of stem and ovary of wild type muskmelon material TopMark,but no obvious expression was detected in the same part of short vine dwarf melon material M406.The results of sequence polymorphism analysis of CmSI gene in different melon materials showed that the mutation of Cmsi only existed in the dwarf mutant M406.The expression analysis of CmSI gene in muskmelon varieties with different plant height showed that the expression level of CmSI gene was positively correlated with plant height.(4)Ectopic overexpression of CmSI gene in Arabidopsis homologous gene mutant er 105 and wild type Col showed that the height of transgenic plants in Arabidopsis increased significantly;similarly,overexpression of CmSI gene in cucumber wild type 3546 led to the increase of cucumber plant height,indicating that CmSI gene plays a positive regulatory role in the development of cucumber and Arabidopsis stem vines.(5)Transcriptome sequencing showed that a large number of auxin transport and signal transduction genes were differentially expressed in the dwarf mutant M406,including members of the ABCB family and several well-known auxin transporter genes in the PIN gene family.The results of hormone content determination showed that the auxin content in the stem of the dwarfing mutant M406 decreased significantly,indicating that the short vine dwarfing of muskmelon may be caused by the decrease of auxin content in the stem.(6)The yeast two-hybrid results of auxin-related genes differentially expressed in CmSI and parent stem vines showed that CmSI protein only directly interacted with CmPIN2,the polar output protein of auxin.Moreover,CmPIN2 can interact with CmΔKinase,the kinase domain of CmSI protein,but not with Cmsi mutant protein.The above results suggest that CmSI may affect the auxin content in muskmelon stems by regulating the expression of CmPIN2,and then regulate the development of muskmelon stems. |
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