The Correlation Analysis Of Keycomponents In Cell Wall Andsaccharification Efficiency Andpreliminary Study Of LcCOMT Bytransformation In Leymus Chinensis

Leymus chinenis is a C3 perennial plant in grass family,which is known as the "king of grass herbage" because it is rich in cellulose,hemicellulose,soluble protein and other nutrients that can be digested and absorbed by herbivorous livestock.Leymus chinenis is the dominant forage and ecological grass in china,it has the characteristics of cold resistance,saline-alkaline tolerance,barren tolerance,and grazing tolerance,and has important economic and ecological values.It has been found that cell wall lignin of forage is an important limiting factor for digestion and absorption of nutrients in livestock.Therefore,it is an important research direction to clarify the cell wall structure and components of Leymus chinensis to genetically modify lignin and improve the digestibility of dry matter by genetic engineering.Here,we analyzed and confirmed the correlation between the components of Leymus chinensis cell wall and saccharification efficiency.Meanwhile,the full length of CDS of COMT,a key enzyme gene for lignin synthesis of Leymus chinensis,was cloned and analyzed by bioinformatics.Efficient in vitro regeneration system and genetic transformation system of Leymus chinensis were established,the overexpression vector and RNAi vector of key enzyme gene COMT for lignin synthesis of leymus chinensis were constructed.The genetic transformation of Leymus chinensis callus mediated by agrobacterium tumefaciens was studied with superexpression vector and RNAi vector.The main results were as follows:1.The correlation between the key components of Leymus chinensis cell wall and saccharification efficiencyThe content changes of three major components of Leymus chinensis cell wall were systematically analyzed.It was found that cellulose and hemicellulose reached the peak at the early developmental stage and remained stable with internode maturity,while lignin content and monomer composition increased significantly in internode during the whole jointing stage.In order to clarify the pathway of lignin synthesis in Leymus chinensis,partial CDSfragments of 10 lignin biosynthesis-related enzymes of Leymus chinensis(CCR,COMT,OMT,PAL,CAD,C3 H,HCT,C4 H,4CL and F5H)were successfully cloned,with the sequence similarity of more than 90% to homologous genes in Aegilops tauschii,Hordeum vulgare and wheat.The expression levels of six genes(PAL,4CL,HCT,C3 H CCo AOMT and COMT)increased gradually with stem maturation at jointing stage,following the model of internode cell wall lignification,and the five genes(PAL,C3 H CCo AOMT,COMT and CAD)remained stable at heading stage with the maturation of stem nodes,which was not consistent with the model of lignin accumulation in the growth of internode.The saccharification efficiency of different tissues is different,with the highest in the leaves,and the lowest in the internodes.Furthermore,the saccharification efficiency is negatively correlated with the total lignin,S and G monomer lignin and the ratio of S/G.However,the content of cellulose and xylose are not associated with saccharification efficiency.2.Cloning of LcCOMT gene in Leymus chinenis and functional validation in vitroLcCOMT gene was cloned with the molecular weight of 38.068 k Da and the isoelectric point of 5.62,the gene ORF span of 1 056 bp and encoding 354 amino acids.The LcCOMT shares high similarity with the homologous gene in Hordeum vulgare and Aegilops tauschii at protein level which are 96.11% and 95.00%,respectively.The protein of LcCOMT is hydrophobic with two conserved domains of dimerization domain and O-Methyltransferase domain,but no signal peptide and transmembrane domain.LcCOMT was expressed in leaves,leaf sheaths,and stem nodes,and with the highest expression in stems.Moreover,the expression of the same stem segment is enhanced along with the elongation.In addition,the gene was also induced by salt and the highest expression was achieved at 200 m M Na Cl for 4 hours.We further performed the enzyme reaction in vitro using the purification recombinant COMT protein.The COMT has a high activity of converting the substrate of caffeoyl-Co A into the product of feruloyl-Co A.3.Establishment of genetic transformation system of Leymus chinenis and the preliminary research of LcCOMT gene transformationThe method of high efficiency germination of Leymus chinenis seeds was established,and the germination rate reached 68.3%;Then,the high efficient regeneration system of Leymus chinenis in vitro was generated,the callus induced from spikelet reached more than 90%,the growth of callus was 58.3%,and the regeneration rate was more than 40%.Finally,the efficient genetic transformation system of Leymus chinensis was established,and the transformation rate reached more than 18%.Next,LcCOMT overexpression(p ANIC-6B-OE-LcCOMT)and RNAi expression vectors(p ANIC-8B-RNAi-LcCOMT)were constructed,genetic transformation of Leymus chinenis was preliminarily confirmed positive by PCR examination and GUS staning,and the functional verification is ongoing.