Screening And Functional Verification Of Key Genes And Non-coding RNA For Eucalyptus Grandis Cellulose Synthesis

Cellulose is an essential structural component in plant cell wall and a renewable biomass resource,which plays a very important role in nature.Eucalyptus(including Eucalyptus grandis)is an excellent timber tree species,which provides a large amount of raw materials for paper industry and wood industry.Synthesis of cellulose is a very complex process which involves a series of important biological processes,each of which is involved by multiple genes and regulated by networks.However,there is still a lack of research on the regulation of related genes and non-coding RNA in the process of cellulose synthesis in E.grandis.In this study,the wood anatomical characteristics and chemical indexes of E.grandis were studied by taking the main stem of three different parts(DBH,middle and upper part of the trunk)of E.grandis as the material.By means of transcriptomics,genomics,molecular biology and bioinformatics,key genes related to the regulation of E.grandis wood cellulose biosynthesis were screened.The EXP,HEX and Su Sy gene families closely related to cellulose synthesis of E.grandis were identified and screened,and the basic characteristics of their gene families were deeply analyzed.Moreover,the regulatory role of non-coding RNA(lnc RNA,mi RNA and circ RNA)on candidate genes was clarified,and the network map of ce RNA regulation during E.grandis wood cellulose biosynthesis was constructed,in order to further explore the molecular regulation mechanism of eucalyptus synthesis of cellulose lay the foundation.The main findings were as follows:(1)The fiber length in the different stem part of E.grandis was different.The average diameter of the fibers in the upper part of the trunk was greater than that in the DBH,and the average diameter in the middle of the trunk was the smallest.The fiber length-width ratio ranged from 34.09 to 82.41,and the average fiber length-width ratio in the middle of the trunk was the largest.The cell wall thickness tended to decrease first and then increase with height,and the cell wall thickness was thinnest in the middle of the trunk.The overall trend of the ratio of cavity diameter was decrease from from the pith to the outside.The overall change trend of luminal diameter ratio gradually decreased from the pith to the outside.The height of wood ray varies between 297.91—448.81 ?m,and the average height of wood ray at the DBH was the smallest,and the average height in the middle of the trunk was the largest.The width of wood ray decreases with the increase of height.The wood ray tissue ratio increased with the height of E.grandis.The contents of cellulose,lignin and hemicellulose were 48.7%—52.9%,15.48%—20.75% and17.15% — 24.75% respectively.The content of cellulose in the middle of the trunk was significantly higher than that at the DBH and the upper part of the trunk.(2)The transcriptome sequencing of 9 samples from the immature xylem at the DBH,middle and upper part of the E.grandis trunk obtained a total of 95.81 G clean reading,57 480 transcripts,7 365 lnc RNA and 5 180 circ RNA.Each sample was identified to obtain 172—306known mi RNA and 1 644—3 508 new mi RNA.A total of 296 differentially expressed genes(DEGs),190 DE-lnc RNA,174 DE-mi RNA and 270 DE-circ RNA were obtained by comparing the expression levels of each transcript.By analyzing the differentially expressed genes,19 key candidate genes,including Ces A5,EXP1,HEX4,Su Sy2,UGT76F1 and UGD1,5 MYB,13 WRKY and 10 NAC transcription factors,5 lnc RNA and 9 mi RNA were screened out and the ce RNA regulatory network was constructed.(3)Based on the transcriptome sequencing results,35 Egr EXP gene family members were identified and screened from E.grandis genome by bioinformatics method,which were distributed on 8 chromosomes.The Egr EXP coding proteins were all located on the cytoplasmic membrane,and 28 of the family members had signal peptides.The proteins which were encoded by the Egr EXP genes were composed by alpha helix,extending chain,random coil and beta corner.10 Egr HEX gene family numbers were identified and screened.The Egr HEX genes were distributed on 5 chromosomes,and the subcellular localization were all on the cytoplasmic membrane.In the Egr HEX gene family,there were significant differences in the structure of each gene,and the number of exons was 7—15.18 Egr Su Sy gene family members were identified and screened,which were distributed on 7 chromosomes.Egr Su Sy proteins were all located on the cytoplasmic membrane,while none of the Egr Su Sy gene family members had signal peptides.There were significant differences in the expression patterns of Egr EXP,Egr HEX and Egr Su Sy genes in immature xylem,mature leaves,phloem,stem tip,xylem and young leaves.(4)Through transcriptome sequencing and gene family analysis,Egr EXP17 and Egr HEX2 were identified and screened,which was closely related to cellulose biosynthesis of E.grandis.Egr EXP17 and Egr HEX2 were overexpressed in 84 K poplar,and the content of cellulose and lignin in transgenic plants were significantly higher than that of wild type 84 K poplar,the average plant height and root number of transgenic plants were significantly higher than that of wild type,and the average diameter in the middle and basal part of the stem were significantly higher than that of wild type.Lnc RNA1 and lnc RNA4 were screened which regulating the genes related to the synthesis of cellulose in E.grandis,and lnc RNA1 and lnc RNA4 were overexpressed in 84 K poplar.The content of cellulose and lignin in lnc RNA4 were significantly higher than that of wild type 84 K poplar and lnc RNA1-oe.The average plant height,the middle and basal part of the stem diameter of lnc RNA4-oe were significantly higher than that of wild type,and there was no significant difference in the growth of lnc RNA1-oe compared with wild type.Here,the cellulose synthesis related genes and non-coding RNA were studied in E.grandis.Those results will provide basis for comprehensive research on the molecular regulation of cellulose synthesis in different species,enriched the research on non-coding RNA in woody plants,and also provide theoretical significance and potential application value for eucalyptus genetic improvement and molecular breeding.