Junjie Bai

Expression of common carp growth hormone in the yeast Pichia pastoris and growth stimulation of juvenile tilapia (Oreochromis niloticus)

Abstract The cDNA encoding mature growth hormone (GH) was cloned from the pituitary of common carp ( Cyprinus carpio ) by RT-PCR. To construct the expression plasmid, the GH cDNA was inserted into the pUC18 plasmid and subsequently subcloned into vector pPICZαA, which contains the promotor from the alcohol oxidase ( AOX1 ) gene and the α-factor signal peptide sequence. The yeast Pichia pastoris GS115 strain was transformed with the expression plasmid. Transgene expression was observed after screening of the transformants with Zeocin™. Results showed that, with methanol induction, recombinant carp GH (rcGH) had been expressed and exported into the culture medium. The production peaked at 72 h of induction and the optimal pH for expression was 6.0. The yield was 300–400 mg l −1 in shaking-flask fermentation medium, accounting for 34.61% of the total supernatant secreted proteins. rcGH was separated through anion-exchange chromatography. The growth rate of juvenile tilapia ( Oreochromis niloticus ) injected with purified rcGH, was found to be 24.5% and 53.1% higher than the control at the dose of 0.10 and 1 μg g −1 body weight week −1 respectively ( P

Validation of reference genes of grass carp Ctenopharyngodon idellus for the normalization of quantitative real-time PCR.

Expression of four reference genes of grass carp, including β-actin (ACTB), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 18S rRNA (18S) and elongation factor-1 alpha (EF1α), was studied in tissues of normal individuals and bacteria-infected individuals. EF1α had the most stable expressions followed by 18S rRNA then GAPDH; ACTB had the least stability. After being infected with bacteria, the grass carp showed minimal changes in expression levels of EF1α in the liver and head kidney, while ACTB had the most stable expressions in spleen but the least stable in liver. EF1α is thus the optimal reference gene in quantitative real-time PCR analysis to quantitate the expression levels of target genes in tissues of grass carp.

Shoaling and mate choice of wild-type Tanichthys albonubes in the presence of the red fluorescent transgenic conspecifics

Shoaling and sexual behaviour of wild-type male and female white cloud mountain minnow Tanichthys albonubes were measured in the presence of the red fluorescent transgenic conspecifics under laboratory conditions. Wild-type female test fish showed no significant preference, whereas wild-type male test fish preferred to be near a shoal of red transgenic fish rather than wild-type fish. When placed in a potentially reproductive context, wild-type males had a higher competitive ability over transgenic males; wild-type females spent more time with wild-type males in visually mediated experiments, but wild-type males performed more courtship displays towards transgenic females. These results suggest that the red body colouration does not appear to disturb signal communication between wild-type and transgenic T. albonubes in shoaling behaviour; transgenic males have no mating advantage over wild-type males, but the red body colouration of transgenic females may affect mate choice of wild-type males.

Complete mitochondrial genome of the green sunfish (Lepomis cyanellus).

Abstract The green sunfish (Lepomis cyanellus) is a species of freshwater fish in the Centrarchidae family of order Perciformes, which is native to a wide area of North America. This species was introduced into China in 1998 and became one of the economically important freshwater species. In this study, we determined the complete mitochondrial genome sequence of L. cyanellus. The total length of L. cyanellus mitogenome is 16,485 bp, which consists of 13 protein-coding genes, 22 tRNA genes, 2 rRNA genes, and 1 control region (D-loop), with the genome organization and gene order being identical to that of the typical vertebrate. The overall base compositions of the heavy strand are 27.5% A, 26.2% T, 29.9% C, and 16.4% G, with an A + T content of 53.7%. The mitochondrial genome of L. cyanellus shared 86.8% sequence identity with that of Lepomis macrochirus. The sequence information presented here will be useful for evolutionary as well as population genetic studied.

Genetic variability and relationships among six grass carp Ctenopharyngodon idella populations in China estimated using EST-SNP Markers

The grass carp, which is one of the most important farmed fish species in China, is widely distributed in different river basins. In this study, 21 single nucleotide polymorphism markers, developed in the grass carp expressed sequence tag database, were used with the snapshot genotyping method to analyze the genetic structure of six grass carp populations (SS, JL, HH, YJ, NX, and FS) from the Yangtze River and the Pearl River. The effective numbers of alleles (Ne) of the six grass carp populations was 1.7680–1.8038, the observed heterozygosity (Ho) was 0.5938–0.6305, the expected heterozygosity (He) was 0.4420–0.4443, and the polymorphic information content (PIC) was 0.3241–0.3403. The PIC, Ne, and He of the JL population were the highest among all populations, whereas those of the YJ population were the lowest. The NJ tree analysis based on genetic distance of the grass carp populations revealed that the six populations clustered into two branches: the SS, JL, and HH populations clustered first into one branch, which then clustered with YJ population; the FS and NX populations constituted a separate branch. The established genetic structures and phylogenetic relationships of some of the grass carp populations of the Yangtze River and Pearl River may lay the foundation for selecting core breeding populations.

Cloning, tissue distribution and effects of fasting on pituitary adenylate cyclase-activating polypeptide in largemouth bass

Pituitary adenylate cyclase activating polypeptide (PACAP) has a wide range of biological functions. We cloned the full-length cDNAs encoding PACAP and PACAP-related peptide (PRP) from the brain of largemouth bass (Micropterus salmoides) and used real-time quantitative PCR to detect PRP-PACAP mRNA expression. The PRP-PACAP cDNA has two variants expressed via alternative splicing: a long form, which encodes both PRP and PACAP, and a short form, which encodes only PACAP. Sequence analysis results are consistent with a higher conservation of PACAP than PRP peptide sequences. The expression of PACAP-long and PACAP-short transcripts was highest in the forebrain, followed by the medulla, midbrain, pituitary, stomach, cerebellum, intestine, and kidney; however, these transcripts were either absent or were weakly expressed in the muscle, spleen, gill, heart, fatty tissue, and liver. The level of PACAP-short transcript expression was significantly higher than expression of the long transcript in the forebrain, cerebella, pituitary and intestine, but lower than that of the long transcript in the stomach. PACAP-long and PACAP-short transcripts were first detected at the blastula stage of embryogenesis, and the level of expression increased markedly between the muscular contraction stage and 3 d post hatch (dph). The expression of PACAP-long and PACAP-short transcripts decreased significantly in the brain following 4 d fasting compared with the control diet group. The down-regulation effect was enhanced as fasting continued. Conversely, expression levels increased significantly after 3 d of re-feeding. Our results suggest that PRP-PACAP acts as an important factor in appetite regulation in largemouth bass.

Characterization of the complete mitochondrial genome of Pelteobagrus intermedius (Nichols et Pope)

Abstract Pelteobagrus intermedius is a unique freshwater fish native species in the Hainan Island and Guangxi and other water systems of China. In this study, the complete mitochondrial genome sequence of P. intermedius was determined from whole genome Illumina sequencing data. The total length of the mtDNA was 16,532 bp, including 13 protein-coding genes, 22 transfer RNA genes, two ribosomal RNA genes, and a non-coding control region. Phylogenetic analysis suggested that the P. intermedius is closely related to Tachysurus fulvidraco. The results will serve as a helpful reference for further studies on the conservation genetics of genus Pelteobagrus.

Polymorohisms of SNPs in ALDO B gene and association analysis with growth traits in grass carp(Ctenopharyngodon idellus)

Aldolase is a glycolytic enzyme,which plays a key and pivotal role in glycolysis and glucose metabolism.And compared to aldolase A and aldolase C,aldolase B is involved in both glycolysis and gluconeogenesis.In this research,the two genomic sequence fragments are amplified according to two contigs cDNA fragments of ALDOB gene from EST database of grass carp(Ctenopharyngodon idellus).Then three nucleotides mutations were identified by using directly sequencing and blasting.The polymor-phisms are C+687G,A117C and C+1042A,which are in exon 6,intron 7 and exon 8 respectively.C+687G is synonymous mutation and-A63C is non-synonymous mutation.296 grass carps were used to genotype polymorphisms and then calculate the genotype and gene frequency by SnaPshot method.Frequencies of the AA genotype were 42.9%,32.8% and 32.8% respectively,the AB genotype were 42.9%,45.9% and 45.6% respectively,the BB genotype were 14.2%,21.3% and 21.6% respectively.A general linear model was used to statistically analyze the relationship between ALDOB gene polymorphisms and major growth traits.The result indicated that the three different genotypes of C+687G site only showed significant association at body length/caudal peduncle ratio(P0.05),while they showed no significant association at body weight.The three different genotypes of A117C andC+1042A sites showed significant association at body weight and so on(P0.05).Three combinations of seven,three and seven diplotypes,with the minor allelic frequencies of above 3%,were constructed based on three SNPs in the experiment population.As-sociation analysis indicated that three combinations of diplotypes were all significant association between diplotypes and body weight(P0.05): the seven diplotypes of C+687G,A117C and C+687G,C+1042A showed significant association at body weight,body width and so on(P0.05).And the three diplotypes of A117C and-43C,C+1042A showed significant association at body weight and space.Not only different genotypes of three SNPs sites,but also diplotypes showed significant association in major growth traits in grass carp.And the homozygous genotypes were higher than heterozygote genotypes in the main growth traits.As a result,ALDO B gene could be a candidate modifier gene for grass carp growth traits which could be useful for grass carp molecular breeding.