Expression Analysis And Functional Verification Of ARF Genes Related To Triploid Loquat Flower Development

Loquat（Eriobotrya japonica Lind.）belongs to the Rosaceae genus,Eriobotrya,and is also one of the important economic fruit trees in south China.The flower,leaf and fruit of loquat can be made full use.Owing to the low edible rate of many seeds in loquat fruit,the cultivation of triploid seedless loquat is one of the major tasks in loquat breeding.However,the fertility of triploid loquat was lower than that of diploid loquat.The pollen of triploid loquat was aborted and could not be pollinated and fertilized normally.So far,there is little research on the molecular mechanism of the fertility of triploid loquat.Auxin is one of the endogenous hormones that promote plant growth and development.It is involved in various stages of plant growth and development,and plays an important role in reproductive processes such as flower development and seed development.Auxin response factors（ARFs）play important roles in auxin signal transductionby directly combining with specific response elements in the promoter of auxin response gene,and then regulate various growth and development processes of plants.In the present study,we focused on the molecular mechanism of flower development related ARFs in regulating the fertility of triploid loquat,which is of great significance for the sexual reproduction of triploid loquat and its further application.In this study,we used the diploid loquat Longquan No.1（B2）and triploid loquat（B355）lines as the research materials.Based on the observation of in situ germination of pollen tube after cross pollination of B355 and the transcriptome sequencing of B355,we cloned the fertility related ARF genes from loquat.Using bioinformatics methods we analyzed the biological characteristics of these ARFs.We examined the the spatiotemporal expression pattern of these genes in diploid and triploid loquat by q RT-PCR.We constructed ARF-GFP transient expression vectors and detected the subcellular localization of EjARF6 and EjARF8.Finally,we genenerated the ectopic expression Arabidopsis lines for EjARF6 and EjARF8 genes,and preliminarily analyzed the role of these two genes in the regulation of fertility.The major findings are as follows:1.Bioinformatics analysis of EjARF8-like1、EjARF8-like2 and EjARF6On the basis of transcriptome sequencing and digital expression profile analysis of diploid and triploid loquat,three ARF-like genes were cloned from loquat,named as EjARF8-like1、EjARF8-like2 and EjARF6.The length of EjARF8-like1 gene is 2424 bp,including a 2178 bp ORF,encoding 725 amino acid residues,with a predicted molecular weight of 80.49 k D and an isoelectric point of 6.29.The EjARF8-like2 gene is 2633 bp long,contains a 2370 bp ORF,encodes 789 amino acid residues,and has a predicted molecular weight of 88.01 k D with an isoelectric point of 5.98.EjARF6 is 2899 bp long,contains a 2694 bp ORF,encodes 897 amino acid residues,and has a predicted molecular weight of 99.34 k D,isoelectric point 6.16.According to homologous comparison and evolutionary tree analysis,EjARF8-like1 and EjARF8-like2 genes are most closely related to At ARF8 in Arabidopsis Thaliana,and highly homologous to ARF8 gene in apple,pear and other species.2.Bioinformatics analysis of proteins encoded by EjARF8-like1、EjARF8-like2 and EjARF6We predicted the secondary structure and tertiary structure,transmembrane region analysis and subcellular localization of the proteins encoded by EjARF8-like1、EjARF8-like2 and EjARF6 genes by bioinformatics methods.It was speculated that EjARF8-like1、EjARF8-like2 and EjARF6 proteins all had conserved B3 domain and Auxin-response domain.The tertiary structures of EjARF8-like1 and EjARF8-like2proteins are very similar to the auxin response factor 1 in Arabidopsis Thaliana.The tertiary structure of EjARF6 protein is very similar to the auxin response factor 5 of Arabidopsis Thaliana.3.Spatiotemporal expression pattern of EjARF8-like1、EjARF8-like2 and EjARF6We detected the spatiotemporal expression pattern of EjARF8-like1、EjARF8-like2and EjARF6 genes in diploid loquat longquan 1（B2）and triploid loquat B355 by QRT-PCR.The results showed that the expression of these genes in diploid loquat was higher than that in triploid loquat.Before pollination,the expression level of these genes decreased successively in present bud stage（a）,microdew stage（b）,dew stage（c）and immediate opening stage（d）.All these genes were expressed in loquat fruit,leaf,root,filament,petal,anther,calyx,pistil and ovary,and the strongest signal was detected in the pistil of the flower.The expression level of these three genes in ovary and calyx is about the same,and the expression level in filaments is lower than that in ovary and higher than that in petals.In loquat bud and diploid loquat treated with hormones and inhibitors at the immediate opening stage,the gene was positively induced by 6-BA and negatively induced by BR,while the response to ABA,GA3,NAA and other hormones,as well as NPA and TIBA inhibitors was noobviously changed.In triploid loquat,the gene was positively induced by ABA,GA3 and 6-BA,and negatively induced by BR and NAA.There was no significant change in NPA and TIBA treatment.The results indicate that the expression level of these genes may be regulated by various plant hormones and has a certain role in the regulation of flower development by plant hormones.4.EjARF8-like1、EjARF8-like2 and EjARF6 Subcellular localization and transcriptional activity analysispBI2300-35s-egfp-EjARF8-like1,p BI2300-35s-egfp-EjARF8-like2,p BI2300-35s-egf p-ejarf6 plant expression vector and p GBKT7 transcription factor activity vector were successfully constructed.Subcellular localization of these EjARFs was predicted in the nucleus,and the localization of EjARF6 was further confirmed by fusion with green fluorescent protein.5.Genetic transformation and functional analysisWe constructed the plgn-35s-EjARF8-like1,plgn-35s-EjARF8-like2 and plgn-35s-EjARF6 plant expression vectors,and transformed these vectors into Arabidopsis Thaliana by agrobacterium-mediated methods.We found that the transgenic Arabidopsis lines exhibited larger leaf,earlier bolting,and deformed flowers.The stamens of some transgenic lines were much shorter than the wild-type stigmmens,and the amount of pollen in these lines was very few.Using I₂-KI pollen staining,we confirmed that the fertility of these pollens was poor,resullting in the seed numbers of these lines were significantly reduced.The hypocotyl length of EjARF8-like1 and EjARF8-like2 transgenic lines was shorter than that of the wild type,while the root length was significantly longer than that of wild type,and the number of lateral roots was more than that of wild type.The length of hypocotyl and root of EjARF6 transgenic lines were significantly longer than that of the wild type,and the number of lateral roots was also more than that of the wild type.These results indicated that EjARF8-like1、EjARF8-like2 and EjARF6 genes could promote plant growth,root growth and lateral root generation.EjARF8-like1 gene is also involved in the regulation of flower development.According to the phenotype of the transgenic Arabidopsis Thaliana,it can be speculated that the genes might be involved in the fertility regulation of loquat.