Molecular Cloning And Expression Analysis Of Lignin Biosynthesis-related Genes In Mulberry (Morus Spp.)

As a biological resource with good adaptability and rich nutrition,forage mulberry can alleviate the contradiction between the rapid development of animal husbandry and the shortage of forage in China.However,due to the presence of lignin in the cell wall,the digestibility of the feed is affected,and the nutrient value is reduced.In order to find out the differences of lignin content and expression of key enzyme genes in lignin biosynthesis in mulberry leaves,and we hope to provide reference for the breeding mulberry of low lignin content.We cloned two key genes in the lignin biosynthesis pathway from mulberry,COMT and CCoAOMT.And we analyzed the expression of these two genes and other three key genes CAD,CCR,F5 H of lignin metabolism among different species,tissues,and stages using real-time quantitative PCR,the total lignin content of mulberry leaves in different strains and different periods was determined.The main research contents of this study on the genes are as follows:1、Cloning and sequence analysis of caffeic acid O-methyltransferase(MmCOMT)gene in mulberry(Morus multicaulis)Caffeic acid O-methyltransferase is a bifunctional methyl transferase that catalyzes the methylation of C3 and C5 in hydroxyl cinnamomum aromatic ring,then produce acacia and erucic acids,and it plays an important role in the control of lignin synthesis.The gene of caffeic acid O-methyltransferase was cloned from young leaves of Yu 71-1(Morus multicaulis),which was designated as MmCOMT(GenBank NO.MK779971).The coding region of this gene is 1125 bp,which encodes a protein of 374 amino acid,and it is compared with the sequences of 8 other plants in the NCBI nucleic acid and protein database.The results show that it has the highest sequence similarity with Morus notabilis(over 94%)and over 68% with other species.2、 Cloning,sequence analysis and prokaryotic expression of caffeoyl-CoA O-methyltransferase(MmCCoAOMT)gene in mulberry(Morus multicaulis)Caffeoyl-CoA O-methyltransferase is a SAM-dependent methyl transferase,it can catalyzes the formation of acacyl CoA from caffeyl CoA,which is one of the key enzymes in lignin synthesis,and it plays an important role in the synthesis of lignin.The gene of Caffeoyl-CoA O-methyltransferase was cloned from young leaves of Yu 71-1(Morus multicaulis),which was designated as MmCCoAOMT(GenBank NO.MK673764).The coding region of this gene is 744 bp,which encodes a protein of 247 amino acid,and it is compared with the sequences of 9 other plants in the NCBI nucleic acid and protein database.The results show that the sequences are highly similar(over 87%).3、 Expression difference of CAD、 CCR、 F5H、 COMT、 CCoAOMT gene in different mulberry strains、tissues、harvest stages and the analysis of lignin relative contentThe results of real-time quantitative PCR and the determination of total lignin content in mulberry showed that the expression levels of those genes were different among different mulberry strains,and the gene expression between different tissues was randomness.The gene expression was generally high in the third harvest period of forage mulberry,and the gene expression was low in the second harvest period.And there was no significant difference in lignin content between different strains.However,the lignin content of 101,111 and fengchi mulberry reached the highest in the third harvest period,and 13,94 and 112 reached the lowest in the third harvest period.The results may provide some references for the breeding of new forage mulberry varieties with low content of lignin.