Identification Of GRAS And ARF Genes Involved In Roots Development In Brassica Napus L.

As an important organ of plants,roots determine the absorbtion and transport of water and nutrients required for plant growth and development,thereby it is important for crop production.Brassica napus(B.napus)is the most important oil crop in China.Its roots belong to tarproot syetem,but the primary root is short and the root system is generally un-developd.The GRAS and ARF transcription factor families are two plant-specific transcription factor gene families.Members of which play key regulating roles in root development.For example,in Arabidopsis,SCR and SHR genes of GRAS gene family were involved in the asymmetric division of root endothelial cells;whereas AtARF8 of ARF gene family regulated root growth.Hence,the identification of the candidate B.napus GRAS and ARF genes involved in root development are important for further analyzing their molecular mechanism in regulating B.napus root development and improving the nutrient use efficiency and yield of B.napus.In this study,we firstly performed a genome-wide identification of the GRAS and ARF gene family members in B.napus genome.Then the sequence characteristics,phylogenetic,linear relationships and protein interactions of candidates were systematically analyzed.The candidates involved in B.napus root development and stress-resistance were further screened based on the RNA-seq and qRT-PCR analyses.The results of this study will support for further study on the molecular mechanism of candidate GRAS and ARF genes involved in regulating B.napus roots development.The main results of this study are as follows:1.Genome-wide analysis of GRAS gene family in B.napus and identification of candidates involved in root developmentWe identified 90 GRAS genes(BnGRASs)in B.napus genome and classified them into 13 subfamilies by phylogenetic analysis.Except for four BnGRASs genes in the A_nn subgenome and 13 genes in the C_nn subgenome whose chromosome locations were unclear yet,the remainings were distributed on the 19 B.napus chromosomes.Sequence feature analysis revealed that 60%of the candidate BnGRASs did not have intron insertions,and 24 motifs were found at the N-terminal of candidate proteins.The GRAS domains at the C-terminal were less conserved among different subfamilies but were highly conserved within each subfamily.The genomic heterogeneous doubling was the main force for the large expansion of GRAS genes in B.napus genome which tended to retain the genes originated from B.rapa.Interacting protein prediction analyses showed that a total of 889 pairs of interaction proteins were predicted,the primary of which were transcription factors involved in transcriptional regulation and signal transduction.RNA-Seq analysis demonstrated that most BnGRASs were widely expressed in different tissues/organs at different stages in B.napus and 25 BnGRASs were highly and/or specifically expressed in roots.RNA-Seq analyses showed that some BnGRASs genes were up-regulated or down-regulated by hormone inductions.qRT-PCR analysis proved that three BnGRASs belonging to SCR and LISCL subfamilies have potentially roles in root stress response.Integrated comparative analysis of GRAS genes across 26 species of algae,moss,fern,gymnosperm and angiosperm specieses revealed that this gene family originated in early mosses and was classified into 19 subfamilies,14 of which may have originated prior to bryophyte.miRNA prediction analyses revealed that a highly conserved miRNA 171 target was observed in the HAM subfamily across land plants,indicating that this kind of miRNA may have important and conservative regulation role for GRAS gene functions.2.Genome-wide analysis of ARF gene family in B.napus and identification of genes involved in root developmentAuxin response factor(ARF)is a member of the plant-specific B3 DNA binding superfamily.Sixty-seven ARF genes were identified in B.napus(BnARFs)genome and were divided into four subfamilies(I–IV).Sixty-one BnARFs were distributed on all chromosomes except C_n02 chromosome;the locations of the remainings were unclear yet(A_n and C_n).Sequence feature analyses revealed that the full length of the BnARF proteins was highly conserved,especially within each subfamily,with all members sharing the N-terminal DNA binding domain(DBD)and the middle region(MR),and most contained the C-terminal dimerization domain(PBI).Twenty-one members had a glutamine-rich MR that may be act as an activator.Accordingly,the intron patterns are highly conserved in each subfamily or clade,especially in the DBD and PBI domains.miRNA prediction analysis revealed that several members in subfamily III are potential targets for miRNA167/160.Many putative cis-elements involved in phytohormones,light signaling responses,and biotic/abiotic stress were identified in candidate BnARFs promoters,implying their possible roles.Interacting protein prediction analyses showed that most BnARF proteins are likely to interact with auxin/indole-3-acetic acid(Aux/IAA)-related proteins,and members from different subfamilies generally shared many common interaction proteins.Whole genome-wide duplication(WGD)by hybridization between Brassica rapa and Brassica oleracea,and segmental duplication mainly led to the large ARF gene expansion in B.napus genome.Gene loss following WGD was biased,with the A_n-subgenome retaining more ancestral genes than the C_n-subgenome.Candidate BnARFs have wide expression profiles across vegetative and reproductive organs during different developmental stages,with 30 genes were highly expressed in roots.No obvious expression bias was observed between A_n-and C_n-subgenomes.Most synteny-pair genes had similar expression patterns,indicating their functional redundancy.RNA-Seq analysis indicated that BnARFs were sensitive to exogenous IAA and 6-BA treatments,especially subfamily III members.qRT-PCR analysis confirmed that 12 BnARFs were up-regulated or down-regulated under exogenous IAA and 6-BA treatments,suggesting they may play an important roles in B.napus roots development and hormone response.