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Molecular Cloning,Expression Profile And Function Analyisis Of MADS-box Genes In Hevea Brasiliensis

Posted on:2018-10-06Degree:MasterType:Thesis
Country:ChinaCandidate:Y J WangFull Text:PDF
GTID:2310330515486889Subject:Biochemistry and Molecular Biology
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Hevea brasiliensis,known as rubber tree,it is an important economic crop in China's tropical region.The natural rubber produced by rubber trees is an important strategic material relating to the national economy and people's livelihood.As the domestic consumption of natural rubber has increased year by year,the natural rubber production is difficult to improve by expanding the scale of planting because of the narrow suitable environment.Improving the level of rubber tree per unit yield by breeding the new rubber tree high yield varieties is one of the effective ways to alleviate the contradiction between the domestic natural rubber production and consumption.However,as a kind of perennial tree,the rubber tree has a long cycle of sexuality.Breeding new varieties of rubber tree are diffcult due to the lower fruit rate,which results in many hybrid combinations matched failed and makes the efficiency of rubber tree selection greatly limited.Therefore,intensive study of the mechanism of rubber tree flower,reproductive development and the fruit setting rate improvement can not only provide a theoretical basis and technical means to elite hybrid configuration for rubber tree,but provide theoretical basis for rubber tree breeding.During the plant floral organs and reproductive development,MADS-box genes are class of important transcription factor family genes and they play a key regulatory role in plant growth and signal transduction:regulating flowering time,determining floral organ development,affecting fruit ripening,regulating the development of vegetative organs and responding to external signals.However,up to now,the species,expression profiles,and detailed characteristics of the MADS-box genes in the rubber tree are still unknown.In this study,several MADS-box genes were identified by transcriptome sequencing of rubber tree floral organs at different developmental stages.The MADS-box genes were further studied by bioinformatics analysis,phylogenetic tree analysis,temporal and spatial expression pattern analysis,subcellular localization as well as transgenic functional validation.The results are as follows:1?The early development inflorescences,stamens and pistils of the rubber tree were sequenced using the Ilumina Hiseq,and 16.23 Gb data were measured.After assembly and de-redundancy,109 411 unigenes were obtained,the average length was 1044 bp,the N50 was 1805 bp and the GC content was 40.68%.These unigenes were annotated at seven functional databases.The results of NR annotation showed that these unigenes had the highest similarity with Jatropha carcas.GO function annotation shows that there were 75 135 unigenes involved in the biological process function,with 47 900 unigenes performing cell composition functions,43 235 unigenes performing molecular functions.KEGG annotation showed that these unigenes were involved in many biological pathways,including carbohydrate transport and metabolism.Membrane transport metabolism and energy metabolism pathways,suggested that carbohydrate synthesis and transport-related genes may play an important role in the development of rubber tree during reproductive development.2?According to transcriptome sequencing results,the 75 candidates of HbMADS-box genes were examined by CDS and Inter-ProScan to confirm the presence of the MADS-box domain.After removing the redundant sequences,a total of 26 non-redundant HbMADS-box genes with complete ORFs were identified in rubber tree and submitted to the NCBI database.Including 6 previously reported HbMADS-box TFs(HbMADSl,HbMADS2,HbMADS3,HbMADS4,HbMADS5,and HbMADS27)and 20 new HbMADS-box TF genes(designated as HbSEP,HbAGL9.1,HbAGL9.2,HbCMBl,HbCMBl-L,HbAGL6,HbAGL8,HbAPl,HbAGL12,HbAG,HbAGL11,HbAGL15,HbSVP1,HbSVP2,HbTT16,HbDEFL,HbPMADS2,HbAGL30,HbAGL61 and HbAGL65).3?Analyzing the sequences of 26 HbMADS-box genes,the results showed that the ORF length distribution of these HbMADS-box genes ranged from 609bp(HbAGL12)-1116bp(HbAGL30),and the amino acid range of the HbMADS-box gene was between 202-371.The corresponding molecular weight distribution ranged from 23.09-41.86 kDa,and the speculated isoelectric point ranged from 6.06(HbSVP2)-9.34(HbAGL11).Sequence alignment results showed that the sequence identity of the protein of MADS-box gene was between 13.67%and 98.57%,and the average agreement was 39.99%.The HbMADS4 and HbSVP1 protein sequences were the most consistent,reaching 98.5%,and the HbAGL30 and HbMADS3 protein sequences were the smallest,only 25%.4?Analyzing the conserved region of HbMADS-box genes,the results showed that all of the 26 HbMADS-box genes contained a highly conserved MADS-box domain with a length of about 50 amino acid residues,a conserved K domain with a distinctly basic helix,a conserved I domain and a very nonconservative C domain.These HbMADS-box genes obtained above are a typical MADS-box gene family.Further analysis of the conserved regions of the 26 HbMADS-box genes encoding proteins showed that in the MADS-box domain,3(R),17(R),20(T),23-24(KR),27(G),30-31(KK),34(E),38-39(LC),48(F),and 52(G)were highly conserved.5?Analyzing the structural characteristics of 26 HbMADS-box genes,the results showed that the HbMADS-box genes usually contained multiple introns(excepted HbAGL61),and the most of HbMADS-box genes contained 5-7 introns,up to 11 introns.Phylogenetic analysis of 26 HbMADS-box genes,the results showed that 26 HbMADS-box genes were divided into three evolutionary branches which included 10 subfamilies(SEP,AGL6,FUL/API,AGL12,AG,SOC1,AGL15,AGL17,SVP,BS and AP3/P1).6?Analyzing the expression patterns of 26 HbMADS-box genes,the results showed that the most of HbMADS-box genes had been found were involved primarily in floral organ specification and inflorescence development.For example,compared with other tissues,HbSEP,HbAGL9.1,HbAGL9.2,HbCMBl,HbCMB1-L,HbAGL6,HbAG,HbDEFL and HbPADS2 were significantly expressed in shoot tip and flower organs;HbAGL15,HbTT16,HbMADS2 and HbAG were mainly expressed in fruit.These results suggested that these HbMADS-box genes may be involved in the floral organ and reproductive development of Hevea brasiliensis.7?Subcellular localization and functional verification of the HbAG gene is highly expressed in the floral organ development of Hevea brasiliensis,which results showed that the HbAG positioning within the nucleus.Transgenic results showed that overexpression of HbAG could shorten plants,shorten the time of vegetative growth,and promote early flowering time.Furthermore,the expression of endogenous genes related to flower development in transgenic Arabidopsis was detected.So overexpression of HbAG in transgenic Arabidopsis plants may put forward the promotion of AtFT,AtAP1,AtLFY and AtFUL genes,AtSVP gene expression and inhibit the AtSVP gene expression.This suggests that HbAG may influence the reproductive development of rubber trees by interacting with these genes.
Keywords/Search Tags:Hevea brasiliensis, MADS-box genes, Floral development, Transcriptome, Gene clone, Functional Verification
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