High Level Expression of Flounder Growth Hormone in Transformed Chlorella and Functional Analysis with Ornamental fish

Abstract

Fast growth is one of the most important factors in reducing generation periods to develop new ornamental fish breeds. However, unlike farmed fish, attempts to promote the growth of ornamental fish using interspecies fish growth hormones (GHs) have been sluggish. For application of interspecies GHs in ornamental fish, flounder (Paralichthys oliveaceus) growth hormone (fGH), the most studies have been conducted, was chosen for verification of the fish GH homology and growth-promoting effect evaluation in ornamental fish. The homology of ornamental fish and flounder GHs was evaluated at the level of in silico and in vivo in this study. Analysis of amino acid sequences showed that five conserved domains (GD1 to GD5) for formation of common features for general folding of GHs and binding with GH receptors were identified. A four-helix bundle structure (up-up-down-down topology) folding pattern was also identified through putative tertiary structure prediction. The fGH was expressed in Chlorella vulgaris PKVL7422 through homologous recombination between nitrate reductase (NR) gene and [CaMV 35S promoter-gene of fGH (codon-optimized for C. vulgaris)-terminator of RBCS2 gene] fragment. The fGH was expressed stably in C. vulgaris PKVL7422 and the expression level was quantified to be 156.273 mg · L-1. Silver butterfly koi (Cyprinus carpio var.flavipinnis C.V) and discus fish (Symphysodon discus) were used as models for growth-promoting effect evaluation. Experimental group and control group of each fish species were fed with feed supplemented with transformed C. vulgaris PKVL7422 (final concentration of fGH was 1.28 mg fGH · kg-1 feed) and wild type C. vulgaris PKVL7422 for 8 weeks. The gap of weight and standard-length growth between the experimental group and control group of each fish species increased as the experimental period increased. The average weight and standard-length of the experimental group were [26.907 ± 2.867 and 27.471 ± 4.47-fold (silver butterfly koi)] and [23.856 ± 3.408 and 32.330 ± 5.115-fold (discus fish)] higher than the control group, respectively. The weight-specific growth rate (SGR; growth % / day) and feed conversion ratio (FCR; wet weight gain / dry feed intake) indices of the experimental group were [1.553 ± 0.032 and 0.189 ± 0.008 (silver butterfly koi)] and [3.753 ± 0.048 and 0.458 ± 0.025 (discus fish)], respectively and the SGR / FCR indices were higher than the control group.