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| Cloning and Expression Analysis of the Laccase Genes from Kiwifruit (Actinidia deliciosa cv. Miliang-1) |
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Abstract Laccases are multi-copper containing glycoproteins. By catalyzation of various kinds of substrates through the oxidation-reduction reaction of copper ions in their conserved domains, Laccases involve in various physiological and biochemical processes, such as lignin synthesization, iron metabolism, flavonoid biosynthesis, wound healing, stress responses and et al. However, their functions in the post-harvest storage have not been reported. In order to study the function of the laccase genes in the kiwifruit storage process, two laccase genes (AdLAC3 and AdLAC7) were cloned from the Actinidia deliciosa cv. Miliang-1 using reverse transcription PCR(RT-PCR) and RACE-PCR methods. The full-length sequences of AdLAC3 were 2594 bp. It contained a 2205-bp open reading frame, encoding a 734 aa polypeptide and its accession number was MF405444. The full-length sequences of AdLAC7 were 2126 bp. It contained a 1695-bp open reading frame, encoding a 564 aa polypeptide and its accession number was MF405447. In addition, two alternative spliced variants (AdLAC3-variant1 and AdLAC3-variant2) of AdLAC3 were obtained. The accession number of AdLAC3-variant1 was MF405445, and the accession number of AdLAC3-variant2 was MF405446. Protein domain analysis showed that AdLAC3 and AdLAC7 both contained three typical copper binding domains (Cu-oxidase_3, Cu-oxidase and Cu-oxidase_2), but AdLAC3 and AdLAC7 were distributed to different evolutionary branching in the phylogenetic tree, which was due to the great difference in their sequences and implied that they may originate from different ancestral genes. Both AdLAC3 and AdLAC7 harbored five introns and six exons. qRT-PCR analysis in different tissues showed that AdLAC3 exhibited the highest expression levels in roots, followed by stems and leaves, but almost no expression level was detected in fruits. Similarly, AdLAC7 gene also showed the maximum expression in roots, followed by stems, while almost no expression was observed in the leaves and fruits. qRT-PCR analysis in the fruits treated with different storage conditions showed that AdLAC3 gene was significantly induced in the fruits either exposing to 25 ℃, 4 ℃or ABA, while the transcriptional level of AdLAC7 gene in fruits sharply increased only under ABA treatment. This suggested that AdLAC3 and AdLAC7 gene played distinct roles in the post-harvest storage of kiwifruit. This study provides new insights for the kiwifruit research in regard to post-harvest storages and quality regulating.
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Received: 22 August 2017
Published: 01 January 2018
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