Identification Of MCTP Genes In Cotton (Gossypium Hirsutum) And Regulation Study Of Arabidopsis MCTP7 Gene

MCTPs(Multiple C2 domain and transmembrane region proteins)containing multiple C2 domains and transmembrane domains,can serve as signaling molecules to mediate the transport of other regulatory factors.Although some studies have shown that MCTPs play an important role in the growth and development of animals and plants,but the function of them in cotton has not been reported.In this study,we identified 33 GhMCTP genes in the genome of G.hirsutum and divided them into 6 subgroups(MCTP I-VI).Furtherly,we analyzed the interaction patterns of GhMCTPs with some mobile proteins,and studied the function of GhMCTPs in the development of cotton meristems by virus-induced gene silencing(VIGS).In addition,the role of MCTP7 in the primary root elongation of Arabidopsis thaliana were analyzed.The main results were summarized as follows:1.Isolation and identification of GhMCTP Proteinwe used the protein sequence of MCTPs in Arabidopsis as a query to search for putative GhMCTPs using BLAST program in the COTTONGEN,and identified a total of 33 GhMCTP members,These GhMCTP family members can be divided into six subgroups(A to F)based on phylogenetic analysis.And through phylogenetic analysis,it was found that most GhMCTPs are more closely related to Arabidopsis.Analysis of the gene structure of MCTP family members revealed that there are thirty GhMCTP genes without introns,and we found that the GhMCTP proteins in the same subgroup contain similar structures and the same number of C2 domains.2.Expression analysis of GhMCTPs gene in various tissues and fiber development stages of upland cottonTo study the function of the cotton MCTPs gene,transcriptome data were used to analyze its expression levels in different tissues under normal conditions.The results showed that GhMCTPs gene was expressed in each tissues and GhMCTP2 was specifically expressed in the petals.In addition,most of the GhMCTPs genes are highly expressed in the ovules of pre-fiber development,among them,GhMCTP3 and GhMCTP9 are highly expressed in 20 days and 5 days of fiber.At the same time,the results of qRT-PCR showed that the expression of most GhMCTPs in the apical buds was higher.3.Analysis of interaction patterns between GhMCTPs protein and mobile proteins and studies of their functionIt has been previously reported that the GhMCTP family acts as a signaling molecule to mediate the transport of other regulatory factors.Therefore,we selected some mobile proteins and GhMCTPs to explore whether GhMCTP proteins interact with these mobile proteins.Yeast two-hybrid assay showed that both GhMCTP5 and GhMCTP12 could interact with TTG3,KNAT1,WUS1 and SFT.GhMCTP6 and GhMCTP 17 could interact with KNAT1 and TTG3,and GhMCTP9 interacted with TTG3 and SFT,it indicated that GhMCTPs may be involved in the epidermis and shoot apical meristem development of cotton.VIGS technology was used to explore the function of each GhMCTPs in cotton growth and development.The results showed that down-regulation of GhMCTP5,GhMCTP7,GhMCTP11,GhMCTP 12 and GhMCTP 17 genes resulted in a shorter plant height,while silencing of GhMCTP9 resulted in higher plants.In addition,after co-silencing of GhMCTP12 and GhMCTP17,the leaves of the plants appeared shrink and Roll down,it was further proved that GhMCTPs are involved in the development of cotton shoot apical meristem.4.Functional study of MCTP7To study the biological function of the Arabidopsis MCTP7 gene,we analyzed the expression pattern of the MCTP7 gene.The results of qRT-PCR showed that MCTP7 gene was expressed in all tissues of Arabidopsis,and the highest expression is in the young root.We obtained the MCTP7 knock out mutant,observing its phenotype,we found that the primary root length of mctp7 mutant was significantly shorter than the wild type and the number of cells in the root apical meristem of mctp7 mutant was less than that of the wild type,further indicating that MCTP7 regulates the growth and development of Arabidopsis root apical meristem.5.Study on the regulation mechanism of MCTP7To explore the regulator of the MCTP7 gene,we obtained that transcription factor PLT2 could binds to the MCTP7 promoter by yeast one-hybrid screening assay.In addition,we demonstrated that PLT2 protein is involved in the regulation of MCTP7 by dual luciferase transcriptional activation experiments.qRT-PCR analysis showed that the expression level of MCTP7 gene was significantly down-regulated in the plt1-4plt2-2 double mutant,and up-regulated in PLT2 overexpressing Arabidopsis,suggesting that PLT2 transcription factor act as a positive regulator for the expression of the MCTP7.We obtained DR5:GUS/mctp7,GUS staining showed that The mctp7 mutants showed down-regulated expression of DR5,indicate that the auxin content in the root tip of Arabidopsis thaliana was significantly reduced.Therefore,it is concluded that the phenotype of shorter primary root length is due to the loss of MCTP7 function,which may leads to the decrease of auxin content in the root tip of Arabidopsis thaliana,which inhibits cell division,eventually reduce the number of cells in the root apical meristem.Through yeast two-hybrid screening experiments,we find that the transcription factor ZML2 can interact with MCTP7,and the relationship between ZML2 and Arabidopsis primary root elongation can be further explored6.Functional redundancy analysis between MCTP7,MCTP4 and MCTP5MCTP4,MCTP5 and MCTP7 are both expressed in the root apical meristem,in order to explore whether there is functional redundancy between them,we identified their double mutants,and we find that the primary root length of the mctp4mctp5 and mctp4mctp7 double mutant was significantly shorter than that of the wild type.At the same time,we used the dual luciferase transcriptional activation assay proved that PLT2 can also bind to the promoters of MCTP4 and MCTP5.Therefore,we conclude that there is functional redundancy between MCTP4,MCTP5 and MCTP7.