Identification Of Kenaf NAC,CesA/Csl,COMT Gene Families And Expression Analysis Under Salt Stress

Kenaf（Hibiscus cannabinus L.）belongs to the genus hibiscus of the Malvaceae family.It’s an annual herbaceous bast fiber crop.Soil salinization is a global ecological and environmental problem that seriously affects all stages of plant growth and seed germination.NAC transcription factor is a unique class of transcription factors in plants and plays an important role in plant growth and stress resistance.Lignin and cellulose are important components of plant secondary cell walls.Lignin synthesis is mainly controlled by the NAC regulatory network.In this study,the salt-tolerant kenaf variety Fuhong 18 and the salt-sensitive variety Zanyin 1 were used as experimental materials.The Hc NAC genes,Hc COMT genes and Hc Ces A genes were identified by bioinformatics combined with kenaf transcriptome screening.The expression patterns of Hc NAC genes and lignin and cellulose synthesis genes under salt stress were analyzed.In addition,the SSR marker sites in the exons of key genes in response to salt stress in kenaf were developed to assist the breeding of new salt-tolerant kenaf varieties through molecular biological means.The important roles of NAC transcription factor in gene transcription regulation and response to stress have been extensively studied in Arabidopsis.However,the genome-wide identification of the kenaf NAC gene family and the expression patterns under salt stress have not been disclosed.The main findings of this study are as follows:1.Genome-wide identification of kenaf NAC gene family and its expression analysis in response to salt stress.Based on the kenaf genome database,a total of 239 Hc NAC genes were identified,which were distributed on 18 chromosomes,and were named Hc NAC001-239according to their positional relationships on the chromosomes.The phylogenetic tree showed that some NAC proteins of kenaf and Arabidopsis had high homology,and were divided into 18 subfamilies（UN,At NAC3,ATAF,NAP,ANAC001,SENU5,TERN,ONAC022,NAC1,NAM,NAC2,Os NAC8,ANAC011,TIP,Os NAC7,ONAC003,ANAC063,ONAC1）.Fluorescence quantitative PCR was used to detect the tissue-specific expression of 13 Hc NAC genes and their relative expression under salt stress.The expression levels of 13 Hc NAC genes showed different trends in different tissues under salt stress.Except for Hc NAC100 and Hc NAC89,which were down-regulated in stems,the remaining 11 genes were up-regulated.In addition,the increase of peroxidase activity in kenaf leaves may also promote the up-regulated expression of Hc NAC gene,thereby improving the antioxidant capacity of kenaf under Na Cl stress and effectively alleviating the damage of reactive oxygen species accumulation to cell membranes.2.Genome-wide identification of kenaf COMT gene family and its expression analysis in response to salt stress.A total of 80 Hc COMT genes were identified by bioinformatics,which were distributed on 15chromosomes.No Hc COMT genes were located on Chr5,Chr12,and Chr18 chromosomes.They were named Hc COMT01-80 according to their chromosomal locations.A phylogenetic tree of 80 kenaf Hc COMT proteins and 14 Arabidopsis At COMT proteins was constructed and divided into 11subfamilies（Group1-11）.q PCR technology was used to detect the tissue expression specificity and relative expression of 6 Hc COMT genes under salt stress.The results showed that under salt stress,the expression levels of Hc COMT12 and Hc COMT27 were down-regulated in stems,and Hc COMT10,Hc COMT11,Hc COMT16,and Hc COMT28 were expressed in stems.The expression level was up-regulated,which affected the lignin deposition in kenaf stems.3.Genome-wide identification of kenaf Ces A/Csl gene family and analysis of their expression in response to salt stress.A total of 57 Hc Ces A genes（Hc Ces A01-57）,distributed on 17 chromosomes（Chr1-17）,were identified in the kenaf genome-wide database using bioinformatics methods.The results of phylogenetic tree cluster analysis showed that the kenaf Hc Ces A gene family can be divided into 8 subfamilies（Uclassified,Csl A,Csl C,Csl B,Csl E,Csl G,Csl D,Ces A）.The Ces A subfamily can be subdivided into six categories P1,P2,P3,S1,S2,S3.Fluorescence quantitative PCR was used to detect the tissue-specific expression of three Hc Ces A genes（Hc Ces A46,Hc Ces A51,Hc Ces A57）and their relative expression under salt stress.The results showed that Hc Ces A46 had a relative expression advantage in the leaves,stems and roots of Zanyin 1;Under salt stress for 14 days,Hc Ces A51 had the same gene expression pattern in leaves,stems and roots of the two cultivars,which were all down-regulated;Hc Ces A57 was up-regulated in leaves and stems of the two cultivars,and down-regulated in roots.4,NAC,COMT,Ces A salt tolerance differential genes encoding protein interaction prediction and gene co-expression analysis.String protein interaction prediction analysis showed that Hc NAC189 interacted with the cellulose synthesis gene Hc Ces A51.Hc NAC023 interacted with ferulic acid-5-hydroxylase（F5H）in the lignin synthesis pathway,which may be related to the expression of lignin genes related.The gene co-expression network showed 10 pairs of genes between Hc NAC and Hc COMT genes had higher co-expression levels than other genes.3 pairs of genes between Hc NAC and Hc Ces A genes had higher co-expression levels than other genes.10 pairs of genes between Hc COMT and Hc Ces A genes.The co-expression levels of the four pairs of genes were higher than other genes,and the expression patterns of each gene pair were similar under salt stress.It is speculated that the kenaf Hc NAC gene may act as an upstream regulatory gene to jointly participate in kenaf lignin and fiber under salt stress conditions.regulation of gene expression.5.Development of SSR markers for key genes in response to salt stress in kenaf.The salt-tolerant cultivar P₁（Fuhong 18）and salt-sensitive cultivar P₂（Zanyin 1）and their hybrid progeny F₁ lines were used as experimental materials.The developed polymorphic molecular markers were used to identify kenaf germplasm resources.Among the 18 pairs of SSR primers,primers Hc SSR06,Hc SSR11,Hc SSR13 and Hc SSR17 had polymorphisms in amplified bands in varieties with different salt tolerance.The primer Hc SSR13 can stably amplify the co-dominant PCR products of the parental parent,distinguish the hybrid from the parental parent,and lay a theoretical foundation for the molecular marker-assisted breeding of new salt-tolerant kenaf varieties.6.Effects of salt stress on the growth of kenaf seedlings and the activities of antioxidant enzymes.Under the stress of 1.2%salt concentration,the fresh weight of P₁,P₂ and F₁ generation plants all showed a downward trend.Under salt stress treatment for 7 days,the growth rate of kenaf seedlings was not significantly inhibited,the plant height was significantly inhibited between 7-14 days,and the overall stem diameter did not change significantly.The cellulose content was between69.43%and 76.59%,and the lignin content was between 15.59%and20.83%.Under the salt stress treatment,the content of both increased gradually without being significantly inhibited.Salt stress led to excessive accumulation of ROS in seedling leaves and caused membrane lipid peroxidation.SOD,POD,CAT activities and MDA content increased with the increase of stress time.In addition,in the process of salt stress,peroxidase activity was similar to lignin and cellulose content changes,and there was a certain correlation.Salt stress may promote the expression of lignin and cellulose synthesis genes by increasing the activity of peroxidase.Increase the content of lignin and cellulose in kenaf stems to resist the harm of adversity stress.