Genome-wide Identification Of Lipoxygenase Gene Family In Poplar And Function Analysis Of PtLOX11

As an important tree species of shelterbelt and timber forest in our country,poplar trees have enormous economic and ecological benefits,as well as unique biological properties of basic scientific interest.However,poplar trees are affected by a variety of environmental stresses,which has led to tremendous economic loss.Improving both the quality and quantity of available poplar wood is one of the major aims of researchers working in this area.In plants,some oxylipins and their derivatives,such as jasmonic acid,leaf aldehydes and divinyl ethers,have been suggested to be involved in plant defense against pests.Biosynthetic pathways that lead to the formation of these oxylipins in plants have been investigated.Such compounds are produced via the lipoxygenase(LOX)pathway.LOXs contribute to plant developmental processes and environmental responses.However,a systematic and comprehensive analysis has not been focused on the LOX gene family in poplar.Therefore,in the present study,we performed a comprehensive analysis of the LOX gene family in poplar,including gene structure,phylogenetic relationship and microarray analysis.Moreover,the evolutionary process of ancestral LOX genes in four modern rosids were studied based on phylogenetic relationship,intraspecies and interspecies gene colinearity.We subsequently study the functional characterization of PtLOX11 by using prokaryotic expression and HPLC prokaryotic expression,CRISPR/Cas9 and overexpression.At last,we carry out CRISPR/Cas9 and overexpression analyses of target genes.The main results as follows:1.Using bioinformatics methods,we identified a total of 20 LOX genes.These LOX genes were clustered into two subfamilies.The gene structure and motif composition of each subfamily were relatively conserved.In each subfamily,the exon/intron structure and motif compositions of the LOXs were highly similar.These genes are distributed unevenly across nine chromosomes.The PtLOX gene family appears to have expanded due to high tandem and low segmental duplication events.A high proportion of LOX genes are distributed preferentially at duplicated blocks,suggesting that tandem duplications contributed significantly to the expansion of this gene family.2.Microarray analysis showed that a number of PtLOX genes have different expression pattern across disparate tissues.We examined the expression patterns of the Populus LOX genes under different stress conditions including infection with Marssonina brunnea,Methyl Jasmonate(MeJA)treatment,and mechanical wounding.The gene expression of most LOX genes is induced or suppressed under these biotic and abioticstresses.3.In this study,we identified 6,20,13,and 11 LOXs in Arabidopsis,Populus,Vitis,and Carica,respectively.Phylogenetic trees are quite informative for obtaining the LOX gene relationships with each other.In this study,the LOX genes are divided into two groups,13-LOX and 9-LOX,consistent with many previous studies.The number of exons in Carica and Populus LOX genes is relatively stable,whereas the exon numbers has changed dramatically in Vitis and Arabidopsis.The MEME server identifies these LOXs all have LOX and PLAT/LH2 domain,and that each sub-family shares a similar motif.4.After interspecies microsynteny analysis,lots of conserved syntenic segments were found,indicating that the strongly conserved microsynteny among these regions across four species is observed significantly.Each rosids specie has its own duplication events except to the W/SGD.In order to improve our understanding on what affects the evolutionary constraints,we measured the Ka/Ks ratios of paralogous pairs of the four species.Amidst all of the pairwise comparison data,only one gene pair,VvLOX6/11 exhibits a Ka/Ks ratio larger than1,suggesting that accelerated evolution with positive selection occurred in this gene pair.5.According to bioinformatics analysis and the expression under MeJA stress by using qPCR technology,we explore PtLOX11 as our target gene.The full-length cDNAs of PtLOX11 were cloned from ‘nanlin 95'.The full length of PtLOX11 is 2643 bp and the deduced protein harbours 880 amino acids.Comparing with the Populus trichocarpa database,there are four different nucleotides and one different amino acid.There are two typical domains Lipoxygenase and PLAT_LH2 are found in PtLOX11 protein.6.The recombinant PtLOX11 protein was obtained by prokaryotic expression.The enzyme activity result show that the high catalytic activity of PtLOX11 protein at 4.5 when the substrate is linoleic acid.The product type of PtLOX11 protein is 9-HPOD by HPLC,so the protein is 9-LOX.The result of subcellular localization of PtLOX11 show that this protein is located in cytoplasm.In conclusion,we performed a comprehensive analysis of the LOX gene family in poplar.We analyze the origin and evolution,gene duplication and loss of LOX gene familes in four rosids species.PtLOX11 gene is isolated followed by subcellular localization analysis,prokaryotic expression and HPLC to research the functional characterization.At last,we carry out overexpression and CRISPR/Cas9 system to analyses the target genes.These results will provide important theoretical significance and application value in revealing the mechanism of PtLOX11 involved in response to abiotic and biotic stress.