Font Size: a A A

Functional Analysis On Cytokinin Transport Of PpABCG14 Transporter In Peach (Prunus Persica)

Posted on:2022-04-01Degree:MasterType:Thesis
Country:ChinaCandidate:X L HuangFull Text:PDF
GTID:2543306806982349Subject:Pomology
Abstract/Summary:PDF Full Text Request
ABC transporters,widely existed in prokaryotes and eukaryotes,are found to participate in transporting a variety of substances,including plant hormone,lipids,heavy metals and their conjugates as well as secondary metabolites,thus play a crucial role during plant growth and development.Cytokinin(CTK)involved in regulating plant growth and fruit development by promoting cell division.And its transport may be mediated by ABC transporter in plant,but the specific transport mechanism remains unclear.Genome-wide identification of ABC transporters in several model plants such as Arabidopsis thaliana,tomato(Solanum lycopersicum)and tobacco(Nicotiana tabacum)have been finished,but genome-wide identification of ABC transporters in peach(Prunus persica)and its role during peach fruit development have not been reported.In the present study,a total of 125 ABC proteins were identified in peach genome database,and phylogenetic relationship,chromosomal location,gene structure and conserved motif,conserved domain and cis-element as well as synteny correlation were also analyzed.The candidate genes involved in the transport of cytokinin were screened based on the response of genes to the treatment of exogenous cytokinin 6-BA in peach.Finally,the function of the candidate gene PpABCG14 was verified by in vitro transport assay in tobacco(Nicotiana tabacum)leaves,atabcg14 mutant complementary experiment and transgenic tomato.These results lay a foundation for further elucidating molecular mechanism of ABC transporters in regulating plant growth and fruit development.The main results are as follows:1.Based on whole genome database,a total of 125 ABC genes were identified in peach.According to phylogenic analysis of ABC genes between peach and Arabidopsis,these peach ABC genes were divided into eight subfamilies,namely,ABCA-ABCG and ABCI.Among 125 genes,ABCG subfamily was the largest subfamily with 56 genes,while ABCD subfamily only had two members.Chromosome location showed that 125 ABC transporters were distributed unevenly on eight chromosomes of peach.A total of24 gene family members were located on the third chromosome,while only the 10 genes were distributed on the seventh chromosome.The analysis of gene structure and conserved motif showed that each subfamily had similar exon-intron structure and conserved motif: each ABC gene had at least one NBD conserved domain.The cis-elements analysis showed that the ABC gene family members contained a large number of plant hormone-responsive elements,which had great potential to play a role in hormone transport.Synteny analysis in peach found four pairs of collinear genes,all of which were derived from whole genome duplication.Synteny analysis among species showed that peach had higher homology with apple,but poor homology with rice.2.The full-length sequence(2031 bp)of Prupe.1G353300 gene was cloned,which encoded 676 amino acids.Phylogenetic analysis showed that PpABCG14 protein was the homologous gene of AtABCG14 and OsABCG18,which belonged to the ABCG subfamily protein and was named as PpABCG14.Secondary structure prediction indicated PpABCG14 was composed of α-helix,random coil and β-extension chain,and accounted for 43.64%,47.93%,8.43%,respectively.According to tertiary structure analysis,PpABCG14 was predicted as the membrane protein and the oligopeptide status was homodimer.The conserved domain analysis showed that PpABCG14 contained one TMD and one NBD,which was a half-size transporter.Based on the full length of the PpABCG14 gene,the overexpression vector p C1300s-PpABCG14-e GFP was constructed.Tobacco transient expression experiment demonstrated PpABCG14 is the plasma membrane-localized ABC transporter and met the basic condition for cytokinin transport.3 The analysis of spatio-temporal expression pattern showed that the candidate genes PpABCG14 mainly expressed in root,leaf,flower and fruit at the early developmental stage,while less expressed in fruit at the late developmental stage.6-BA spraying treatment can significantly increase the fruit weight but remarkably decreased the fruit shape index,which showed that exogenous 6-BA treatment significantly improved the fruit weight by promoting cell division(P < 0.05).PpABCG14 was upregulated in leaves treated with 6-BA for 7 days(DAB 110),and the expression level of PpABCG14 was also significantly increased in peach fruit after leaves were treated with 6-BA for 14 days(DAB 117)(P < 0.05).The results of gene expression and exogenous hormone treatment showed that PpABCG14 may be related to export of cytokinin from root to or stem to young sprout and fruit sink tissue.Therefore,it was speculated that PpABCG14 might play an important role in regulating peach plant growth and fruit development.4.In vitro transport assay,the exported cytokinins,including t Z、t ZR、c ZR、DZR、i PA,were significantly higher in PpABCG14-transformed tobacco leaves than in empty vector transformed tobacco leaves,but there was no significant difference in ABA transport(P < 0.05).In the sodium vanadate inhibition experiment,various cytokinins(tZ、tZR、 cZR、DZR、iPA)in the efflux solution were significantly decreased,while the content of exported ABA showed no significant difference(P < 0.05),which suggested PpABCG14 had the function of cytokinin transport.5.The phenotype complementary experiment of atabcg14 mutant showed that plants phenotype recovered to wild-type plants after overexpressing PpABGC14 gene in atabcg14 mutant,and there were no significant differences in rosette leaves,plant height and number of siliques between the transgenic plants and wild-type plants(P <0.05),indicating that PpABGC14 and At ABCG14 had similar biological function.Meanwhile,ABC gene PpABCG14 was overexpressed in Micro-Tom tomato,and T1 generation seeds of PpABCG14 transgenic tomato were obtained.These results suggested that PpABGC14 played an important role in the transport and distribution of cytokinin in peach and regulated plant growth and fruit development by transporting cytokinin in plant.
Keywords/Search Tags:Peach, Cytokinin, ABC transporter, Growth and Development, Transgenic tomato
PDF Full Text Request
Related items