Molecular Evolution Of Heat Shock Transcription Factor Gene Families In Four Model Angiosperms

With the increase of high temperature,the general reaction of organisms is to produce heat shock proteins,which plays an important role in the reduce production of agricultural and sideline products and the decline in quality.Organisms in the body above the normal growth temperature of 5 °C will be in vivo rapid synthesis of some heat shock proteins,which as the central regulators of the expression of heat shock proteins and other heat shock induced genes,having an important role in improving the thermotolerance of crops.In recent years,studies on the HSF family had been thoroughly characterized in Arabidopsis,rice,poplar,Chinese cabbage,maize,and Malus,whose genomes have been sequenced.This provided a convenient opportunity for us to analyze the evolution and expansion of the HSF gene family in four model angiosperm,namely Populus trichocarpa,Arabidopsis thaliana,Oryza sativa,and Vitis vinifera,for which substantial information about genomes or transcriptomes was available.The goal of this study was to ask whether this model of protein-coding gene family evolution applies to the HSF gene family,and,if so,whether there is an association between the evolution of HSF genes and changes in expression patterns that might indicate diversification of function.Meanwhile,phylogenetic tree analysis,exon/intron organization and motifs,gene duplication,intraspecies / interspcies microsynteny analysis,duplication event dating and environmental selection analysis and gene expression patterns of HSF genes were also investigated to perfect related information about this family.The results of this research were as follows:1.Sequences of Arabidopsis and rice HSFs were downloaded from the Arabidopsis Information Resource(TAIR,http:// www.arabidopsis.org/,release v10.0)and rice annotation database(http://rice.plantbiology.msu.edu/,release v5.0).We performed BLAST(Basic Local Alignment Search Tool algorithms)searches against the poplar and grapevine genome database using 22 of the known Arabidopsis full-length HSF rotein sequences as queries,and then using the resulting poplar and grapevine sequences as secondary queries.All potential candidates were subjected to SMART and Pfam analyses to ensure that the predicted proteins contained consevred domains or sequence motifs.We identified 30 HSF in poplar and 10 HSF in grapevine.2.Phylogenetic analysis showed that the 87 HSF genes from the four model angiosperms could be grouped into three classes that corresponded to the classes A,B,and C.Class A and Class B were subdivided into eight(A1–8)and five(B1–5)clades according to their bootstrap values and phylogenetic relationships.3.We further examined the exon/intron organization of 20 paralogous pairs in these four species to investigate traceable intron gain or loss within these.Most of the closely related genes within the same subfamily shared similar gene structure in terms of either exon lengths or intron numbers.Among these,only two paralogous pairs(Os Hsf-08/-21 and Pt Hsf-16/-20)showed any difference.These differences might be derived from the exon/intron loss or gain event.We used MEME motif detection software to reveal the diversification of HSF genes.Each of the putative motifs was annotated by searching Pfam and SMART.The most common motif was motif 1.4.By searching for intraspecies microsynteny,it shows that the expansion of the HSF gene family is mainly through large-scale duplication events,but not tandem duplication.We identified 18 paralogous gene pairs of duplicated segments resulting from a whole-genome duplication in poplar,Arabidopsis,and rice.Notably no paralogous gene pairs existed in grapevine.5.Using a stepwise gene-by-gene reciprocal comparison of the regions hosting the HSF genes,we observed strongly conserved microsynteny among Arabidopsis,rice,grapevine,and poplar.Therefore,we referred to these 15 distinct groups as orthologous groups.After two rounds of ancient gene duplication events,73.3% of the ancient replication genes in poplar were retained,just 32% in rice and only 9% in Arabidopsis,and all of the genes in grapevine were lost.6.Selection pressure analysis shows that continuous purifying selection has played a major role in the maintenance of HSF genes in angiosperm evolution process.However,there were still some conserved regions of HSFs subjected to positive selection.The results showed that HSF family in angiosperms were under purifying selection,and these genes were under strong evolutionary constraints so as to maintain its stable function.Additionally,a few conserved coding regions in the HSF gene family underwent positive selection,which indicated some new function in these HSF genes.7.Microarray data analysis showed that most of HSF genes in different tissues have obvious specific expression.Among them,there were 7 groups of duplication genes with similar expression patterns,only two groups(Pt Hsf-19/17 and Pt Hsf-23/27)on the expression pattern of gene duplication is slightly different.8.We used qRT-PCR to analyze the expression levels of HSF genes in popalr in response to heat shock treatment,cold stress and oxidative stress.The results showed that most of the HSF genes were induced by these three kinds of stress.Most of the duplicated genes have similar induction patterns.Pt Hsf-10 was significantly up-regulation by heat stress.Therefore,we cloned the full length genes of Pt Hsf-10 and laid a foundation of further study on the function.