Construction Of The Gene Network Of Phosphorus Utilization Pathway In Brassica Napus L.and Screening Key Candicate Genes In It

Phosphorus（Pi）is one of the essential macronutrients for plant growth and development.The application of Pi fertilizer can increase crop yield and quality.Brassica napus L.（B.napus）is one of the main oil crops and edible vegetable oil source in China.B.napus requires a mass of Pi and is very sensitive to Pi level,and a large amount of Pi fertilizer is usually applied in production to ensure crop yield and quality.However,Pi utilization efficiency of B.napus is low,excessive fertilization not only fails to sustain high yields,but also leads to high production costs,waste and even depletion of phosphate rock resources,soil pollution,and eutrophication.Therefore,improving the Pi utilization efficiency is a crucial objective for molecular breeding in B.napus,which is of great significance to increase the yield and efficiency,and sustainable development of B.napus in China.In plants,the Pi utilization efficiency is mainly determined by Pi utilization pathway.To date,the gene networks related to Pi utilization have been well understood in model plant,Arabidopsis and Oryza sativa.However,there is still a lack of systematic research on the Pi utilization pathway in most crops,such as B.napus.In the present study,we firstly identified the Pi utilization related genes at the genome-wide level by Bioinformatics methods,and constructed the gene network of the Pi utilization pathway in B.napus;Then,we carried out a systematic Bioinformatics analysis of this pathway（e.g.,subcellular location,chromosome location,gene duplication mechanism,transcriptional regulating mechanism,expression profile,co-expression network,etc）;Secondly,we performed an in-depth analysis of the PHT gene family that plays a central role in Pi utilization;Finally,based on the above Bioinformatics analysis,we screened 3 candidate genes for Pi utilization,and analyzed their expression mechanism under low Pi stress by qRT-PCR.Together,this study provides essential gene resource for further study on the molecular mechanism of the candidates in Pi utilization in B.napus.The main results of this study are as follows:1.Genome-wide analysis of the Pi utilization related genes in B.napusIn this study,we identified 285 genes of the Pi utilization network in ZS11 genome by Blast P method,including 4 transcription factor（TF）families（MYB,WRKY,SPX and b HLH）and 17 structural gene families,respectively.The subcellular localization analysis showed that the Pi utilization proteins were mainly located in the nucleus（～46.0%）and cell membrane（～36.5%）,whereas the rest were primarily located in chloroplast（～7.7%）,vacuole（～2.8%）or mitochondria（～3.5%）.Chromosome localization analysis showed that the Pi utilization related genes were unequally distributed on the 19 chromosomes of B.napus,and they tend to distribute on C_n-subgenome（A_n:131,C_n:149）.Collinearity analysis showed that expansion of Pi utilization related genes were mainly attributed to small-scale duplication events:including segmental duplication（69 genes,～26.9%）,homologous exchange（58 genes,～22.6%）and segmental exchange（20 genes,～7.8%）events.Meanwhile,the allopolyploid（78 genes,～30.4%）between Brassica rapa and Brassica oleracea also plays a key role in gene expansion of Pi utilization pathway in B.napus.Notably,the genes derived from B.rapa genome were tended to be duplicated and retained in B.napus.Transcription factor（TF）binding site prediction showed that Pi utilization-related genes are widely regulated by 362 TFs belonging to 32 TF families.Mi RNA regulatory site prediction revealed that 56 types of mi RNAs might have 305potential regulatory sites in 90 Pi utilization related genes.Interaction protein prediction analyses suggested that 67 pairs of interaction proteins were predicted for 33 Pi utilization proteins in B.napus,indicating that the Pi utilization proteins tend to form protein complexes to act their functions.Spatio-temporal expression analysis demonstrated that most Pi utilization-related genes were widely expressed during the whole developmental stages in B.napus,and the expression trends of structural genes and TFs are different,indicating their different roles in Pi utilization process in B.napus.Moreover,most of the synteny-gene pairs（106,79.1%）may be functional redundancy while the rest（28,20.9%）may undergo neo-functionalization or sub-functionalization during the evolution.Furthermore,low Pi stress expression profile analysis showed that90（～31.58%）Pi utilization-related genes were differentially expressed in root and/or leaf tissues under low Pi stress,indicating their potential roles in Pi utilization in B.napus.The co-expression analyses found that the hub genes were genrally the members of Pi transporter（PHT）family,indicating the important roles of PHT genes for Pi utilization in B.napus.2.Genome-wide analysis of PHT gene family in B.napusWe further permormed an in-depth analysis of the PHT gene family in B.napus.Accordingly,we identified 80 PHT genes（Bna PHTs）in B.napus genome.Phylogenetic analyses showed that the PHT family could be divided into five subfamilies（PHT1-5）.Subcellular localization analysis showed that members of the subfamilies were mainly located in the cell membrane（Bna PHT1s,Bna PHT4s）,chloroplast（Bna PHT2s）,mitochondria（Bna PHT3s）and vacuole（Bna PHT5s）.The conserved protein domains analysis showed that the conserved domains of five PHT subfamilies were different,which might related to the different functional characteristics among the five subfamilies.Chromosome localization analysis showed that the Pi utilization related genes tend to distribute in C_n subgenome（A_n:34,C_n:42）.Collinearity analysis showed that small-scale duplication events were the main driving force for large gene expansion of the Bna PHTs,including segmental duplication（33 genes,～41.3%）,homologous exchange（11 genes,～13.8%）and segmental exchange（20 genes,～7.8%）events.Notably,merely 15 genes（～18.8%）were derived from allopolyploid between B.rapa and B.oleracea,which might attribute to a lot of homologous exchange events of Bna PHTs in ZS11 genome.The spatio-temporal expression analysis demonstrated that most of the Bna PHTs have wide expression profiles in B.napus,and the expression patterns within each subfamily or clade were similar.Low Pi stress expression profile analysis showed that 11 Bna PHT1s and four Bna PHT3s were strongly induced（Fold Change≥2,FDR<0.01）by low Pi stress,whereas members of the other subfamilies have no obvious expression change under low Pi condition.3.Screening candidate genes involved in Pi utilization in B.napusBased on the spatio-temporal expression and low Pi stress expression profiles analysis,we screened three candidate Bna PHTs genes（Bna PHT1.34,Bna PHT1.29 and Bna PHT1.7）.Bna PHT1.34 gene has the highest differential expression（log₂FC=7）in leaves under low Pi stress,Bna PHT1.29 gene has higher differential expression（log₂FC=3）in roots under low Pi stress,whereas Bna PHT1.7 gene is differentially expressed in both leaves and roots under low Pi stress.The co-expression analyses of spatio-temporal expression profiles showed there are 43 Pi utilization related genes that constitute 70 pairs of co-expression relationships with the three candidate genes（Pearson correlation coefficient>0.6;P-value≤0.01）.Similarly,there are 32 Pi utilization related genes that constitute 46 pairs of co-expression relationships with the three candidates,indicating their hub roles in Pi utilization gene network.Moreover,qRT-PCR analysis further confirmed the expression patterns of the three candidates in response to low Pi stress by RNA-Seq analyses.