Transcriptome Analysis Of Fiber Development And Identification Of Auxin Signaling Related Genes In Flax(Linum Usitatissimum L.)under High Temperature

Flax is an important global economic crop with excellent characteristics and various commercial uses.It has been widely used as fiber,edible oil,medicine,feed,and composite materials.In recent years,frequent extreme climates(including high temperatures)have occurred frequently,which increasingly threaten the crop production.Flax is a crop suitable for growing in a cool environment and is greatly affected by temperature.Unsuitable growth conditions will significantly affect the anatomical structure and fiber formation of flax stems.However,there are few reports on the molecular mechanism of high-temperature affecting flax fiber development.This study combines next-generation sequencing and full-length transcriptome sequencing techniques to explore the effect of high temperature stress on flax fiber development And on this basis,early response to auxingene families,SAUR and GH3,and auxin transporter gene families,AUX/LAX and PIN,were used for genome-wide identification and expression pattern analysis.The main content and results of this study were as follows:1.Under high temperature stress,samples were took from the bark of the upper,middle,and lower parts of the flax stalk(representing three different periods of fiber development),which were analysed using next-generation transcriptome sequencing and full-length transcriptome sequencing techniques.The numbers of differentially expressed genes in the upper,middle and lower parts of HT stem bark compared to CK were:2889,3131,and 5244,respectively.GO enrichment analysis revealed that HT stress affected the differential expression of genes in the upper part of stem bark,mainly enriched in the biosynthetic pathway of phenylpropane biosynthesis/lignin;the differential expressed genes in the middle part of stem bark were mainly enriched in the hydrolase/galactosidase activity/β-galactosidase activity pathway;differential expressed genes in the lower part of stem bark are mainly related to the synthesis of cellulose synthase.We investigated the differentially expressed genes related to cell wall synthesis.The expression patterns of these genes vary greatly at different stages of fiber development.In general,the expression of CesA,SUS,BGAL,PEL,PAL,CAD,LAC genes were down-regulated,most of XTH,EXP,PME,CCoAOMT genes expression down-regulation.In addition,under HT stress,the auxin-responsive genes SAUR,ARF,AUX/IAA and the auxin transporter AUX/LAX,PIN genes were differentially expressed,and most of the genes were consistently up-regulated or down-regulated at different stages of flax fiber development.Among them,two SAUR genes,four ARF genes,three AUX/IAA genes,one AUX/LAX gene expression were down-regulated;three SAUR genes,three ARF genes,two AUX/IAA genes,one AUX/LAX gene and four PIN genes were up-regulated.This difference ultimately may affect the phloem thickness,fiber cell cross-sectional area,thickness of the fiber cell wall,and the number of the fiber cells were significantly lower than the control.The chemical composition in mature fiber showed that HT stress significantly reduced the content of cellulose and significantly increased the content of pectin.Based on the above analysis and the acid growth theory,we speculated that the early response gene of flax auxin and the auxin transporter gene may play an important role in the resistance of flax to HT stress or in the process of fiber development,so we conducted follow-up research2.Through genome-wide identification and analysis of flax SAUR and GH3 family genes,a total of 86 LusSAUR genes and 10 LusGH3 genes were identified Bioinformatics analysis of LusSAUR and LusGH3 gene families,and through the published gene chip data and the transcriptome data in this study,the expression patterns of these genes in different tissues and HT stress were obtained.Select genes that were relatively highly expressed in bark tissue or respond to HT stress,such as LusSAUR9,12,15,18,35,39,50,72,73,83 and other genes.The gene expression patterns in root,stem and leaf tissues were obtained by qRT-PCR under hormone treatment(IAA,MeJA,ABA,SA)and abiotic stress(PEG,NaCl,CdC12).The results of qRT-PCR showed that in flax seedlings treated by IAA,the genes of LusSAUR,and LusGH3 could be rapidly up-regulated in a short time in response to auxin.In addition,the expression pattern of the LusSAUR gene changed under hormone treatment and abiotic stress,indicating that LusSAUR responded to hormone and environmental signals during plant growth and development.The analysis suggested that the LusSA UR gene might play more important role in regulating flax fiber development and corresponding high temperature stress.This study laid the foundation for further research on the function of early auxin-responsive genes in flax growth and development.3.Through genome-wide identification and analysis of flax AUX/LAX and PIN family genes,a total of 8 LusAUX/LAX genes and 8 LusPIN genes were identified.The LusAUX/LAX and LusPIN genes family were analyzed for bioinformatics analysis,and the expression patterns through the gene chip data of different flax tissues and the transcriptome data in this study were analyzed.It was speculated that the LusAUXla,LusAUX1b,LusLAX1a,LusLAX1b,LusPIN1,and LusPIN3 genes might be involved in the development of flax phloem fibers.LusLAX1b,LusPIN3,LusPIN5a,and LusPIN5b genes responded to HT stress in the stem bark tissue of flax.LusLAX1a,LusLAX1b,LusLAX3b,and other three genes had variable splicing.In order to furtherly study the function of LusAUXla and LusPIN5a genes,the LusAUXla gene was heterologously expressed in Arabidopsis.It was found that the roots of transgenic Arabidopsis seedlings germinated for 7 d were significantly longer than WT,and at the mature stage,the number of cells in the vascular bundles of transgenic Arabidopsis increased.The VIGS technique was used to silence the LusPIN5a gene in flax.Cytological analysis showed that the silencing of the LusPIN5a gene affected the morphology and arrangement of xylem cells,and the phloem fibrous cells showed an uneven size and increased cell numbers.Therefore,by studying the expression patterns of LusA UX/LAX and LusPIN family genes and the function of some genes,high-quality candidate genes were provided for the development of flax phloem fibers.