利用次世代定序來解析日本鰻胚胎、前柳葉鰻、柳葉鰻及玻璃鰻之轉錄體概況並探討其仔稚魚消化和吸收能力

Abstract

近幾年，因為過漁、棲地破壞、海洋環境改變等因素影響，造成日本鰻鰻苗的資源量急遽下降。因此，為了減少對野生資源的利用，發展人工繁殖技術來健全日本鰻的養殖產業已經成為主要的目標。而人工繁殖技術的發展，在育苗的階段因無法開發出仔稚魚合適的飼料轉換技術，使得仔稚魚成長率及存活率偏低。本研究透過與日本研究團隊合作，在馬里亞納海脊以西海域附近採集了3隻日本鰻柳葉鰻、5隻前柳葉鰻、及10顆的胚胎，以及在宜蘭河口採集到1隻玻璃鰻。並藉由次世代定序技術來重建日本鰻完整的轉錄體以及探討仔稚魚消化道裏存在的消化酶和營養運輸蛋白之基因表現情況，進一步釐清日本鰻仔稚魚對食物消化以及營養吸收的能力。 本定序實驗共產生約三億條長度為100 bps的短序列，濾除adaptors及品質較差的短序列之後，再經由序列組裝程式Trinity的組裝，共組出224,043條轉錄序列。其中116,146條序列被預測具有可轉譯區域(長度大於50 a.a.)。而這116,146條序列與蛋白質資料庫nr database進行比對，有70,096條序列可以比對到特定蛋白質。而在這些蛋白質中，有90.2%在輻鰭魚綱中可找到同源基因。而從實驗的分析結果顯示，前柳葉鰻有很好的蛋白質消化能力，但是醣類及脂質的消化能力並不佳。而在柳葉鰻裡也同樣有類似的模式，但是醣類及脂質整體的消化能力比前柳葉鰻還要來的好。另外，玻璃鰻對蛋白質、醣類、脂質的消化能力則都差不多。而在營養吸收方面，在前柳葉鰻和柳葉鰻，的確也有較好的胺基酸吸收能力，而醣類的吸收能力次之，但對膽固醇有較差的吸收能力。而玻璃鰻有極佳的胺基酸吸收能力，醣類吸收能力比起前兩個階段也有下滑的趨勢，有趣的是，膽固醇運輸蛋白則是近乎沒有表現。前柳葉鰻和柳葉鰻腸道的醣類消化能力並不好，但卻有良好的吸收能力，這也許跟腸道益生菌協助消化有關，或者是仔稚魚本身偏好直接吸收食物中的單醣和雙醣。還有玻璃鰻對於脂質的消化能力不差，但膽固醇運輸蛋白表現量低，這也許是跟有良好功能的脂質分解酶和細胞膜本身的組成特性有關，因為小分子的脂質代謝產物通常可以直接穿過細胞膜。以上這些資訊，能提供仔稚魚飼料開發及配方比例轉換技術有利的參考資料。而關於這四個階段的轉錄體序列資料及註解資料，已經建立線上資料庫開放查詢，來協助日本鰻基礎生物研究的進行。

Natural stocks of A. japonica’s glass eels have decreased dramatically due to overfishing, environmental destruction and change of ocean conditions in recent years. Therefore, in order to reduce the consumption of wild glass eels, developing techniques of artificial production for improving Japanese eel farming industry has become a major goal. As a result of no appropriate feed conversion method currently applied on the breeding process of eel larvae, the growth rate and survival rate are extremely low. In this study, three leptocephali, five preleptocephali, and ten embryos were collected near the Mariana ridge by cooperating with the Japanese team, and one glass eel was collected at the estuary of Yilan River. All the samples were submitted for RNA-seq by next-generation sequencing for reconstructing complete transcriptome of Japanese eel. Furthermore, the expressions of digestive enzymes and nutrient transporters existing in small intestine were investigated for realizing the digestive and absorptive capacities of Japanese eel larvae. A total of ~30 million raw reads (100 PE) were generated in our experiment and assembled into 224,043 transcripts after eliminating adaptors and low quality reads. Moreover, 116,146 transcripts were predicted to have open reading frames, and these putative protein-coding transcripts were submitted to blast against nr database. Subsequently, 70,096 transcripts were found positive hits, 90.2% of which were homologous genes of Actinopterygii. Besides, our analytic results showed that the digestive capacity of protein is terrific in preleptocephalus, but the digestive capacities of carbohydrate and lipid are very poor. In addition, a similar pattern is also found in leptocephalus, but the digestive capacities of carbohydrate and lipid are relatively better than in preleptocephalus. Furthermore, glass eel has average capacities for digesting protein, carbohydrate and lipid. In nutrient absorption, the absorptive capacity of amino acids is actually the best in preleptocephalus and leptocephalus, followed by the absorptive capacity of glucose which is better than cholesterol. Additionally, in glass eel, the absorptive capacity of amino acids is also superb, but the absorptive capacity of glucose is significant decreased than previous stages. Interestingly, the cholesterol transporters almost have no expression in glass eel. The digestive capacity of carbohydrate is poor in preleptocephalus and leptocephalus, but absorptive capacity is good. This may be related to gut microbiota in them, or they prefer to directly absorb monosaccharide or disaccharide in the food. Moreover, the digestive capacity of lipid is terrific in glass eel, but the absorptive capacity of cholesterol is poor, which may be associated with the well-functioned lipase and structural properties of plasma membrane because the small molecules of lipid end-products usually can pass through the plasma membrane. Based on above results, much useful information can be provided for developing artificial feed and feed conversion method. An online database for accessing the de novo assembled transcripts of four transcriptome data and annotated result of each transcript has been established. This database will be public for researching community of Japanese eel, and it can assist researchers in studying fundamental eel biology and artificial production.