Cloning and expression analyses of DlLac7 in Dimocarpus longan

Abstract:【Objective】Laccase belongs to a family of multi-copper oxidases that widely distribute in plants, fungi, bacteria and insects. In plants, laccase is involved in lignin biosynthesis, wound healing,stress response, pigment synthesis, and so on. The objective of the current study is to discover the relationship between laccase and longan development and various abiotic stresses.【Methods】Based on the database of longan transcriptome, the c DNA sequences of laccase gene were isolated from longan(‘Honghezi'cultivar) embryogenic callus by reverse transcription-polymerase chain reaction(RT-PCR) and rapid amplification of c DNA end(RACE). Based on the unigene sequence(Unigene68183) of the potential Dl Lac7 in longan transcriptome, two pairs of 5'RACE primers(5P and Dl Lac7-5RACE1, 5NP and Dl Lac7-5RACE2) were designed to amplify the 5'-terminal nucleotide sequence of Dl Lac7, which was 145 bp.In the meantime, two pairs of 3'RACE primers(3P and Dl Lac7-3RACE1, 3NP and Dl Lac7-3RACE2)were designed to amplify the 3'-terminal nucleotide sequence of Dl Lac7, and two different-length sequences(456 bp and 399 bp) were obtained. After fragment assembly, two full-length c DNA sequences were obtained, which were 2 121 bp and 2 064 bp, respectively, and shared a same open reading frame(ORF, 1 713 bp). Based on their 5'-and 3'-terminal nucleotide sequence, a pair of gene specific prim-ers(Dl Lac7-CF1 and Dl Lac7-CR1) was designed to check the full length coding sequence(CDS). Realtime quantitative PCR(q PCR) assay was used to analyze the relative level of gene expression during longan somatic embryogenesis(SE) in different longan tissues, and in response to hormones and abiotic stresses. The process of longan SE contained six stages, including embryogenic callus(EC), incomplete compact pro-embryogenic cultures(ICp EC), globular embryos(GE), heart-shaped embryos(HE), torpedo-shaped embryos(TE), and cotyledon embryos(CE). The samples for longan tissue specific expression analysis of gene included roots(R), leaves(L), floral buds(FB), flowers(F), young fruits(YF), mature fruits(MF), pulps(P), pericarps(PC), and seeds(S). Hormone and abiotic stress treatments included different concentrations(0, 25, 50, 75, and 100 μmol·L^-1) of salicylic acid(SA) and methyl jasmonte(Me JA),salinity(Na Cl, 150 mmol·L^-1), osmotic stress(mannitol, 150 mmol·L^-1), drought(PEG4000, 10%), and abscisic acid(ABA, 10 μmol·L^-1) at different time of treatments(0, 1, 2, 4, 8, 12, 16, and 24 h). The expression level of Dl Lac7 was normalized to reference genes(EF-1a, e IF-4a, and Dl FSD1a), and the data were means ± SD(n= 3).【Results】After verification, two transcripts of longan Dl Lac7 gene(Dl Lac7-a, and DlLac7-b; Genbank number: KM103385 and KM103386) were isolated and the length of the full-length c DNA sequences were 2 121 bp and 2 064 bp, respectively. Dl Lac7-a, and Dl Lac7-b shared an ORF whose length was 1 713 bp and encoded 570 amino acids. The 5'untranslated region(5'UTR) length of DlLac7-a, and Dl Lac7-b were the same, 160 bp, while the 3'untranslated region(3'UTR) length of DlLac7-a, and Dl Lac7-b were different, 248 bp and 191 bp, respectively, including their own poly A, 13 bp and 21 bp, respectively. The amino acid sequence of Dl Lac7 shared highest similarity with that in Citrus sinensis, followed by Populus trichocarpa, and then Vitis vinifera, etc. Bioinformatic analysis revealed that the deduced Dl Lac7 protein contained its conserved domains which shared the typical characteristics of the laccase family. Moreover, Ps RNA Target software was used to predict the potential micro RNA who performed repressive regulation to its target gene by cleavage or translation inhibition mechanism, and the results showed that there might be cleavage site of mi R397 from longan micro RNA database in Dl Lac7 sequence. The q PCR analysis indicated that during longan SE, the level of Dl Lac7 transcripts reached the highest at the stage of the torpedo embryo(TE). Tissue specific expression analysis indicated that Dl Lac7 most abundantly accumulated in longan flowers, followed by flower buds, and then roots, and there was relatively low Dl Lac7 expression in other tissues(leaves, young fruits, mature fruits, pulps, pericarps, and seeds) of longan. After exposure to phytohormones and abiotic stress, expression of Dl Lac7 was induced by SA and a relatively low concentration of Me JA. The salinity, osmotic stress, drought, and ABA stress all gradually induced Dl Lac7 expression from 1 h to 24 h.【Conclusion】These results suggested that DlLac7 might be involved in transcriptional regulation during the middle stages of longan SE and it might participate in the pigment synthesis during longan flowering process. Furthermore, Dl Lac7 might be involved in various kinds of hormone and abiotic stress responsiveness, like jasmonic acid, SA, and ABA,salinity, osmotic stress, and drought stress responses.