Isolation,expression and single nucleotide polymorphisms(SNPs) analysis of LACCASE gene(LkLAC8) from Japanese larch(Larix kaempferi)

摘要

Nucleotide diversity(π) and linkage disequilibrium(LD) analysis based on SNP marker could provide a sound basis for choosing an association analysis method.Japanese larch(Larix kaempferi) is an important timber coniferous tree species for pulping and papermaking,but its high lignin content has significantly restricted it application potential.In this study,the LACCASE gene,that plays an important regulatory role for lignin biosynthesis,was selected as research target.The full-length c DNA and genomic sequences of the encoding Lk LAC8 gene were isolated from the LACCASE expressed sequence tags of the Japanese larch transcriptome database using the rapid amplification of c DNA ends-polymerase chain reaction(RACE-PCR).The c DNA was determined to be 1940 bp,with an open reading frame(ORF,1734 bp) that encoded a protein of 577 AA.This protein contains four highly specific Cu^(2+) binding sites and 11 glycosylation sites,thus belonging to the LACCASE family.The deduced protein sequence shared an 89% identity with the Pta LAC from Pinus taeda.A real-time PCR analysis showed that the Lk LAC8 transcript was expressed predominantly in mature xylem,with moderate levels in the immature xylem,cambium and mature leaves,the lowest in the roots.Lastly,the genomic sequences of Lk LAC8 in 40 individuals from six naturally distributed populations of Japanese larch were amplified,and a total of 201 SNPs(103 and 98 mutation types of transition and transversion,respectively) were detected;the frequency of the SNPs was 1/19 bp.Nucleotide diversity among the six populations ranged from 0.0034 to 0.0053,which suggested that there were no significant differences among the populations.The LD analysis showed that the LD level decayed rapidly within the increasing length of the Lk LAC8 gene.These results implied that LD mapping and association analysis based on candidate gene may be feasible for the marker-assisted breeding of new germplasms with low lignin in Japanese larch.