Juan Tian

Evaluation of reference genes for quantitative real-time RT-PCR analysis of gene expression in Nile tilapia (Oreochromis niloticus).

Quantitative real-time reverse-transcriptase polymerase chain reaction (RT-qPCR) has been used frequently to study gene expression related to fish immunology. In such studies, a stable reference gene should be selected to correct the expression of the target gene. In this study, seven candidate reference genes (glyceraldehyde-3-phosphate dehydrogenase (GADPH), ubiquitin-conjugating enzyme (UBCE), 18S ribosomal RNA (18S rRNA), beta-2-microglobulin (B2M), elongation factor 1 alpha (EF1A), tubulin alpha chain-like (TUBA) and beta actin (ACTB)), were selected to analyze their stability and normalization in seven tissues (liver, spleen, kidney, brain, heart, muscle and intestine) of Nile tilapia (Oreochromis niloticus) challenged with Streptococcus agalactiae or Streptococcus iniae, respectively. The results showed that all the candidate reference genes exhibited tissue-dependent transcriptional variations. With PBS injection as a control, UBCE was the most stable and suitable single reference gene in the intestine, liver, brain, kidney, and spleen after S. iniae infection, and in the liver, kidney, and spleen after S. agalactiae infection. EF1A was the most suitable in heart and muscle after S. iniae or S. agalactiae infection. GADPH was the most suitable gene in intestine and brain after S. agalactiae infection. In normal conditions, UBCE and 18S rRNA were the most stably expressed genes across the various tissues. These results showed that for RT-qPCR analysis of tilapia, selecting two or more reference genes may be more suitable for cross-tissue analysis of gene expression.

Analysis of differential gene expression under low-temperature stress in Nile tilapia (Oreochromis niloticus) using digital gene expression

Tilapia (Oreochromis niloticus) do not survive well at low temperatures. Therefore, improvement of the low-temperature resistance has become an important issue for aquaculture development of tilapia. The objective of this study was to construct a digital gene expression tag profile to identify genes potentially related to low temperature in tilapia. In this study, tilapia was treated at 30°C to lethal temperature 10°C in decrement of 1°CD(-1). Digital gene expression analysis was performed using the Illumina technique to investigate differentially expressed genes in tilapia cultured at different temperatures (30°C, 26°C, 20°C, 16°C, and 10°C). A total of 206,861, 188,082, 185,827, 188,067, and 214,171 distinct tags were obtained by sequencing these five libraries, respectively. Compared with the 30°C library, there were 304, 407, 709, and 772 upregulated genes and 342, 793, 771, and 1466 downregulated genes in 26°C, 20°C, 16°C, and 10°C libraries, respectively. Trend analysis of these differentially expressed genes identified six statistically significant trends. Functional annotation analysis of the differentially expressed genes identified various functions associated with the response to low-temperature stress. When tilapia are subjected to low-temperature stress, expression changes were observed in genes associated with nucleic acid synthesis and metabolism, amino acid metabolism and protein synthesis, lipid and carbohydrate content and types, material transport, apoptosis, and immunity. The differentially expressed genes obtained in this study may provide useful insights to help further understand the effects of low temperature on tilapia.

Dietary available phosphorus requirement of adult GIFT strain of Oreochromis niloticus reared in freshwater

The present study was conducted to estimate the optimum requirement of dietary available phosphorus for adult GIFT strain of Nile tilapia Oreochromis niloticus reared in freshw ater. Six semi-purified diets w ere formulated to contain graded levels( 0. 15%,control diet,0. 40%,0. 66%,0. 91%,1. 17% and1. 43% t) of available phosphorus( KH2PO4w as the source of phosphorus). Each diet w as fed to triplicate groups of 20 fish w ith initial average w eight( 184. 46 ± 8. 13) g for 9 w eeks. The results show ed that fish fed the tw o diets w ith available phosphorus levels of 0. 66% and 0. 91% had significantly higher w eight gain rate( WGR) and specific grow th rate( SGR) than those fed the other diets( P 0. 05),and the feed efficiency( FE) was significantly higher than that of control diet( P 0. 05). The whole body viscerosomatic index( VSI),hepatosomatic index( HIS) and condition factor( CF) decreased significantly with the increases of the dietary available phosphorus levels( P 0. 05),w hile the survival rate( SR) of fish had no significant differences among various dietary treatments( P 0. 05). Crude protein,ash,and phosphorus concentration in w hole body increased significantly w ith the increases of the dietary available phosphorus levels,as w ell as vertebrae and scale ash and phosphorus concentration( P 0. 05). Crude lipid concentration in w hole body decreased significantly w ith the increases of the dietary available phosphorus levels( P 0. 05). Quadratic curve analysis based on SGR indicated that the minimum dietary requirement of available phosphorus for maintaining maximal grow th of tilapia w as 0. 85%.

The changes in growth, serum biochemical indices and GH/IGF-I/IN mRNA expression abundance of Oreochromis niloticus during fasting and re-feeding

Studies were conducted to reveal the changes in growth, serum biochemical indices and growth hormone (GH), insulin-like growth factor Ⅰ(IGF-Ⅰ) and insulin (IN) mRNA expression abundance of Nile tilapia (Oreochromis niloticus) during fasting and re-feeding. Nile tilapia with initial body weight (62.50 ± 3.44) g were starved for 28 d and then fed for 21 d under controllable indoor environment. Fish were sam-pled at 0, 7, 14, 21 and 28 d during fasting and at 14 and 21 d during re-feeding, respectively. The results indicated that the body weight was significantly decreased when fasting over 7 d (P0.05), and signifi-cantly increased when re-feeding for 21 d (P0.05). The hepatosomatic index was significantly decreased throughout the experiment (P0.05). Significant reduction was observed in the content of triglyceride and glucose, and in the activities of alkaline phosphatase, glutamic oxaloacetic transaminase and glutamic py-ruvic transaminase after fasting (P0.05); After re-feeding, the value of these indices increased in varying degrees, but the activity of transaminase was significantly lower than initial value(P0.05). There was no change in total cholesterol, high density lipoprotein cholesterol or low density lipoprotein cholesterol dur-ing the experiment (P0.05). Serum GH and liver GH mRNA levels showed significantly up-regulation, whereas significant down-regulation was observed in serum IGF-Ⅰ and liver IGF-Ⅰ mRNA levels after fasting, and after re-feeding both of them increased (P0.05). IN mRNA level was significantly increased during fasting for 7-21 d (P0.05), but its level was not obviously changed when fasting for 28 d (P0.05), then was significantly decreased after re-feeding (P0.05). The present study revealed that fasting could restrain the growth of O. niloticus, promote serum triglyceride and glucose breakdown, and decrease the activity of transaminase. Serum GH/IGF-Ⅰ and liver GH/IGF-Ⅰ mRNA expression abundance displayed synchronous changes and the liver IN mRNA expression abundance was significantly increased when fasting for 7-21 d and then decreased to normal level when fasting for 28 d. It suggests that the level of GH/IGF-Ⅰ gene transcription of O. niloticus may be the most important factor in determining the levels of hormone in serum, while down-regulation of serum insulin may be due to its release reduction in islet when fasting.