藉yb1與hepcidin基因表現促進魚類生長及免疫功能並鑑別prl 1為新的生長標記

Abstract

由於海洋資源之枯竭，捕撈之魚產自1988年後，呈現停滯不前，全球水產養殖將持續增加，以水產養殖產量增長趨勢與及海洋資源耗竭程度觀之，未來漁業生產的主要驅動力為水產養殖。台灣的水產養殖、種苗生產與水產生物技術優越，但受限於國內水土資源不足，養殖產業規模不但無法擴大發展，而亞太地區每年所需之種苗產值在 10 億美元以上，因此，水產種苗極適合我國養殖漁業作為轉型發展之目標，至目前為止，魚類生長速度、抗病能力仍然是養殖生產急需突破的主要瓶頸之一，若能利用水產生物技術將可加速種魚之育種選拔與及不用抗生素之魚類抗病之方法，增加台灣水產養殖之國際競爭力。 吳郭魚為世界上主要水產養殖魚種之一，亦為台灣重要之水產養殖魚種，育種出快速生長的吳郭魚可提升養殖效率，降低生產成本為吳郭魚養殖產業需解決之問題之一；本研究利用六個分別位於生長激素 (growth hormone, GH)、第二型類胰島素生長因子 (insulin-like growth factor-2, IGF-2)、泌乳激素-1 (prolactin-1, PRL-1)與胰島素 (insulin)等生長相關基因之微衛星DNA (microsatellite DNA)分析鑑別不同生長速度之尼羅種吳郭魚 (Oreochromis niloticus)，研究結果顯示，PRLⅠ-MS01與PRLⅠ-MS02 等兩個位於泌乳激素基因啟動子之微衛星基因標記除可鑑別兩個不同生長速度之尼羅種吳郭魚品系外，其與歐亞種吳郭魚雜交之子代亦可以鑑定其親代，並且發現此生長之差異可能由於泌乳激素基因啟動子含有之微衛星GT或CA重複次數不同，影響基因的表現，而使生長速度不同；除此之外，此兩個微衛星基因標記可同時用以鑑別莫三比克種 (O. mossambicus)、歐亞種 (O. aureus)、賀諾奴種 (O. hornorum)、斯皮路勒種 (O. spilurus)等五種吳郭魚品種。 利用本實驗室自行建立包含吳郭魚肝臟201個EST與肌肉485個EST之晶片 (microarray chip)分析生長快速吳郭魚與腹腔注射GH之吳郭魚肌肉組織，兩組所獲得前十名之ESTs (expression sequence tag, EST)均相同，其中排名第一之基因均為Y-box binding protein 1（yb1），表示此基因具有潛力作為高成長基因標記，選殖吳郭魚yb1基因結果顯示yb1 cDNA全長為1517個nucleotides，推測可轉譯出325個胺基酸，比較吳郭魚與其他魚類yb1之胺基酸序列，其相似度為77-87%，與其他脊椎動物之相似度為46-67%。分析不同餵食條件下之yb1基因表現，結果顯示igf1與yb1 mRNA之表現皆隨著進食之量而增加，禁食使igf1 mRNA停止表現；進一步以顯微注射 (microinjection) yb-1表現載體至斑馬魚一個細胞期 (one-cell stage) 之受精卵，在受精後第一天與第二天，過度表現 yb1之胚胎體節生長 (somatic growth)增加了10-15%，顯示了yb1可能參與了促進魚類生長之功能。 養殖魚類面臨到抗生素濫用所產生之抗生素耐藥性病原菌 (antibiotic-resistent pathogens)，急需找尋新的方法以控制病原菌感染，普遍存在各種生物體之抗菌胜肽 (antimoicrobial peptides, AMPs)即被寄予厚望；本研究由九間波羅肝臟選殖出在許多魚類皆會表現之鐵調素 (AN-hepcidin)，以脂多醣 (lipopolysaccharide, LPS) 誘導AN-hepcidin於九間波羅之組織表現，肝臟、胃、心臟、小腸、鰓與肌肉AN-hepcidin mRNA表現皆顯著增加；以化學合成之九間波羅AN-hepcidin體外測試其對水產病原菌 (aquatic pathogens)之抗菌能力，結果顯示，AN-hepcidin對溶藻弧菌（Vibrio alginolyticus）、腸炎弧菌（Vibrio parahaemolyticus）、創傷弧菌（Vibrio vulnificus）、親水性產氣單胞菌（Aeromonas hydrophila）與無乳鏈球菌（Streptococcus agalactiae）皆具有抗菌活性；以溶藻弧菌與創傷弧菌感染斑馬魚，發現合成之AN-hepcidin可顯著提升斑馬魚之存活率。 綜合上述之結果，位於泌乳激素啟動子之微衛星為一新穎性基因標記，可鑑別快速生長之吳郭魚；由晶片分析結果所發現之yb1基因，可能參與了促進魚類生長之功能，具有潛力用於吳郭魚種苗之分析，以輔助篩選快速生長之吳郭魚；九間波羅之AN-hepcidin扮演重要功能於魚類之先天免疫，可運用於魚類養殖以抵抗病原菌。

Due to the depletion of marine resources, since 1988, catching fish become trapped; global aquaculture production will continue rise. Point of view to aquaculture production growth trends and the degree of depletion of marine resources, main driving force for aquaculture is in fishery production in the future. Taiwan of aquatic farming, seedlings production and aquatic biological technology superior, but by limited to domestic soil and water resources insufficient, farming industry scale not only cannot expand development. Asia-Pacific annual by needed of seedlings output in 1 billion dollars above, so, aquatic seedlings for Taiwan farming fisheries seems as transformation development of target. Currently, fish growth rate and disease-resistant capacity still is farming production needed breakthrough of main bottleneck. If it can use aquatic biotechnology it will be able to accelerate the breeding of fish, selection method of fish disease without antibiotics, and increasing Taiwan international competitiveness of aquaculture. Tilapia is a major aquaculture food fish worldwide. Selection for rapid growth in tilapia is necessary to enhance the competitive advantage of the aquaculture industry. Microsatellites with alternating purine-pyrimidine simple sequence repeats in the promoter have been shown to regulate gene transcription. We found that by examining a combination of two microsatellites located within the proximal promoter of the prolactin 1 (prl 1) gene, PRL I-MS01 and PRL I-MS02, we were able to discriminate between two O. niloticus strains that exhibit distinctive growth traits. There were less GT/CA tandem repeats in the rapidly growing strain N2 than in the slowly growing strain N1. A higher expression level of prl 1 in muscle tissue was observed in the N2 strain in response to growth performance during growth trials. Taken together, more rapidly growing Nile tilapia strains with shorter GT/CA tandem repeat lengths in the prl 1 promoter region and higher prl 1 gene expression levels in the muscle could serve as potential markers for rapidly growing O. niloticus strain selection. In the present study, we investigated the effect of growth hormone treatment on gene expression in tilapia (Oreochromis mossambicus). Microarray analysis revealed increased expression of yb1and igf1 in tilapia treated with growth hormone. We cloned the yb1 gene and found that it encoded a 325 amino acid (35.8 kDa) protein. Sequence identity analysis revealed the tilapia yb1 gene has 77%-87% identity with that of other fishes and 46-67% with that of other mammalian vertebrates. We further investigated yb1 expression in tilapia under different dietary conditions. While expression of both yb1 and igf1 increased with increasing food intake, starvation conditions abolished igf1 but not yb1 expression. This result indicates the activity of yb1 is still required during starvation. To investigate the biological function of yb1, we injected this gene into zebrafish at the one-cell stage. We observed somite growth enhancement of 10-15% in embryos overexpressing yb1. The hepcidin gene is widely expressed in many fish species and functions as an antimicrobial peptide, suggesting that it plays an important role in the innate immune system of fish. In the present study, the Amatitlania nigrofasciata hepcidin gene (AN-hepc) was cloned from the liver and its expression during an immune response was characterized. The results of quantitative PCR and RT-PCR showed that the AN-hepc transcript was most abundant in the liver. The expression of AN-hepc mRNA was significantly increased in the liver, stomach, heart, intestine, gill and muscle but was not significantly altered in the spleen, kidney, brain or skin after lipopolysaccharide challenge. The synthetic AN-hepc peptide showed a wide spectrum of antimicrobial activity in vitro toward gram-positive and gram-negative bacteria. In particular, this peptide demonstrated potent antimicrobial activity against the aquatic pathogens Vibrio alginolyticus, V. parahaemolyticus, V. vulnificus, Aeromonas hydrophila and Streptococcus agalactiae. The in vivo bacterial challenge results demonstrated that the synthetic AN-hepc peptide significantly improved the survival rate of S. agalactiae- and V. vulnificus-infected zebrafish. Taken together, these data indicate an important role for AN-hepc in the innate immunity of A. nigrofasciata and suggest its potential application in aquaculture for increasing resistance to disease. In summary, microsatellite DNA in promoter of prolactin is a new genetic marlers, which can be used to identify the fast-growing tilapia. yb1 found in the analysis of microarray chip may promote the growth of fish. With the potential for analyzing of tilapia fry, yb1 can be used to sort the fast-growing tilapia. AN-hepcidin plays an important role in the innate immunity in fish. It can be used to resist the pathogen in fish farming.