Genome-wide Analysis Of LAC Family In Phyllostachys Edulis And Functional Analyses Of PeLAC And PeLAC190

As one of the important forest resources,bamboo plays an important role in guaranteeing timber safety and ecological security in China.Laccase(LAC),a copper blue oxidase,is involved in lignin monomer polymerization and response to abiotic stresses,which has vital roles in plant growth and development and adaptation to the environment.Moso bamboo(Phyllostachys edulis)was selected as the experimental material in this study.We principally focused on the functional analyses of PeLAC and PeLAC190 based on a genome-wide analysis of LAC family in moso bamboo,which is helpful to provide a reference for bamboo molecular breeding.The main results are as follows:1.Twenty-three homologue genes of LACs were obtained in moso bamboo genome by means of bioinformatics analyses,whose gene structure,physical and chemical properties had significant differences.Phylogenetic analysis showed that these genes belonged to five subfamilies of LAC family,whose intron numbers ranged from 1 to 7,and the number of introns was different even in the same subfamily.The expression patterns of different PeLACs in moso bamboo were analyzed on the basis of the transcriptome data.The result showed that there were significant differences in the expression levels of different genes in root,stem,20 cm shoot,50 cm shoot,early flowering inflorescence and late flowering inflorescence,which suggested that they may played diverse roles in different tissues.2.Two homologue genes of LACs were isolated by the method of homologous cloning and named as PeLAC and PeLAC190.The cDNAs of PeLAC and PeLAC190 were 1 692 bp and 1 719 bp,and the corresponding genomic sequences were 2 785 bp and 2 391 bp,respectively.Both PeLAC and PeLAC190 had six exons separated by five introns,and they all had the targeted site of miR397 based on the analysis of psRNATarget.PeLAC and PeLAC190 encoded 563 and 572 amino acids respectively,they both had the unique structure of LAC,with three conserved domains of copper iron,and 10 histidine residues and 1 cysteinyl residue which were necessary for the activity center of LAC.In addition,the precursor sequence of Phe-miR397(88 bp)was also obtained.3.The cleavage sites in PeLAC and PeLAC190 targeted by Phe-miR397 were validated by RLM-5’ RACE technology,which were located between the 10 th and 11 th bases.PeLAC and PeLAC190 had an opposite expression trend with that of Phe-miR397 in the increasing shoots,which further confirmed that Phe-miR397 could regulate its target genes of PeLAC and PeLAC190 at transcript level.PeLAC and PeLAC190 were constitutively expressed in moso bamboo,both with the highest level in stems,followed by roots.PeLAC and PeLAC190 were up-regulated under the treatments of NaCl(400 mmol·L–1)and ABA(100 μmol·L-1),while it was down-regulated under the treatment of GA3(100 μmol·L-1).PeLAC190 was not obviously but PeLAC was suppressed by the treatment of CuSO4(5 mmol·L-1).These results indicated that PeLAC and PeLAC190 targeted by Phe-miR397 played important roles in hormone signal transduction and response to stresses.4.The over-expression vectors of Phe-miR397 and its target genes of PeLAC and PeLAC190 were constructed,and transferred into the model plant of Arabidopsis thaliana(Col-0)by dipping flower method,respectively.Compared with Col-0,the PeLAC and PeLAC190 transgenic plants were smaller,the petioles were longer,and the lignin content in stems was increased;while the Phe-miR397 transgenic plants had less lignin content of stems.Both PeLAC and PeLAC190 transgenic plants had an increased tolerance to natural drought condition,while that of Phe-miR397 transgenic plants was decreased.The survival rates of transgenic plants overexpressing PeLAC and PeLAC190 were higher than that of Col-0 under phenolic acid treatment.Therefore,the results indicated that PeLAC and PeLAC190 targeted by PhemiR397 participated in lignin synthesis and the abiotic stress response.5.The yeast expression vector harboring PeLAC with own signal peptide was constructed and transformed into Pichia pastortis strain GS115.The positive colonies were verified on the MM plate containing 0.3 mmol·L-1 CuSO4 and 0.2 mmol·L-1 ABTS,which presented dark green after induction of methanol.The positive colonies were induced in liquid medium by methanol,the supernatant was taken and analyzed by SDS-PAGE method.The result showed that there was a specific band at about 77 kDa,which was consistent with the expected fusion protein.In vitro enzyme analysis indicated that the maximum activity of the combinant protein was about 2.031 U·m L-1 after induction for 48 h.For the analyses of the enzymatic reaction,the optimum pH value was 4.0 and the optimum reaction temperature was 40℃.Therefore,it was feasible to obtain the enzyme protein with biological activity by using P.pastortis to express PeLAC.This study provided a reference for further function study of the laccase genes involved in lignin biosynthesis and abiotic stresses in moso bmboo,which also provided new genetic resources for the regulation of plant lignin content and stress tolerance breeding by using genetic engineering methods in the future.