Association Analysis Of Functional Genes And Key Degumming Enzymes In Ramie Degumming Strain Dickeya Dadantii DCE-01

Dickeya dadantii DCE-01,screened and isolated by Institute of Bast Fiber Crops,Chinese Academy of Agricultural Sciences,is an efficient ramie-degumming strain.Utilizing of DCE-01,the gums in ramie raw material could be removed within 8 h.To date,DCE-01 was one of the best biological degumming strains,and has good degumming efficiency and performance on a variety of bast fiber raw materials.Herein,we performed de novo sequencing and assembly of DCE-01 genome.Tandem Mass Tag quantitative proteome of DCE-01 was also analyzed.Then,the key degumming genes and proteins were identified and cloned.Collectively,findings of the current study could advance our understanding of the high-efficient degumming mechanism of DCE-01 strain,and lay a foundation for the exploration of excellent biological degumming strains.The results are as follows:1.The DCE-01 genome was 5.1 Mb in size with 56.64% GC content.A total of 4361 gene models were predicted with an average sequence length of 1005 bp.The overall length of coding genes was about 4.4 Mb.A total of 246 non-coding RNAs were predicted,including 22 r RNAs,148 s RNAs and 76 t RNAs.In DCE-01 genome,21 key degumming enzyme genes of ramie were identified,including 18 pectinase genes,one mannanase gene and two xylanase genes.Then,for the first time,four pectinase genes pel207?pel225?pel G32 and pel027 were cloned in this strain.The genomic sequences of DCE-01 were submitted to NCBI database(Gene number: SRR12328003).2.The expression of proteins during the degumming process of ramie was analysed using TMT-labelled quantitative proteomics.1474 proteins were identified,with 8038 peptides and 242275 secondary spectra,of which 29205 were valid.The total number of differential proteins identified at different degumming time points were as follows: 698 proteins at 8 h-0 h,720 proteins at 16 h-0 h,728 proteins at 24 h-0 h,96 proteins at 16 h-8 h,199 proteins at 24 h-8 h and 199 proteins at 24 h-16 h.The total number of protein differences at 24 h-16 h was 79.3.GO and KEGG enrichment analysis showed that the extracellularly secreted differential proteins of strain DCE-01 have multiple functions and are involved in a variety of metabolic regulatory processes.Cluster analysis of 14 key ramie degumming enzymes showed that Compared to 0 h,1mannanase was down-regulated at 8 h,16 h and 24 h,1 xylanase was down-regulated at 8 h and up-regulated at 16 h and 24 h,2 pectinases were down-regulated at 8 h and 10 up-regulated,3pectinases were down-regulated at 16 h and 9 up-regulated,7 pectinases were down-regulated at 24 h and 5 up-regulated;compared to 8 h,1 mannanase was down-regulated at 16 h and 24 h,1 xylanase was up-regulated at 16 h and 24 h,8 pectinases were up-regulated and 4 down-regulated at 16 h,5pectinases were up-regulated and 9 down-regulated at 24 h;compared to 16 h,1 mannanase was down-regulated at 8 h,16 h and 24 h,1 xylanase was down-regulated at 8 h and up-regulated at 16 h and 24 h.5 pectinases were up-regulated and 7 down-regulated at 24 h.The expression of pectinase plays a major role in the degumming process.4.The prokaryotic expression systems of four pectinase genes were constructed,and the gene sequence information of the four pectinase genes were analyzed and the 3D models of the proteins were predicted.The secondary structures of the four pectinases all had typical ?-helix,extended chain and irregularly coiled structures.The pectase activity of the four recombinant strains p EASY-E1-207,p EASY-E1-225,p EASY-E1-G32 and p EASY-E1-027 were 17.76 U/m L,18.37 U/m L,20.88 U/m L and19.84 U/m L,respectively.