• ISSN 1000-0615
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Volume 9 Issue 11
Nov.  2021
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Effects of Saccharina japonica enzymatic hydrolysis extract on the growth and hepatopancreas transcriptome of Ictalurus punctatus

  • Corresponding author: YE Yuantu, yeyt@suda.edu.cn
  • Received Date: 2020-10-09
    Accepted Date: 2021-01-19
    Available Online: 2021-10-13
  • The aim of this study was to investigate the effects of kelp enzymatic extract on the growth and hepatopancreas transcriptome of Ictalurus punctatus. We prepared different diets with enzymatic extract of kelp addition amount of 0 g/kg (S), 0.3 g/kg (KP3), 0.5 g/kg (KP5), 1.0 g/kg (KP10), 1.5 g/kg (KP15), 2.0 g/kg (KP20) to feed channel catfish in cage-culture for 60 days. Channel catfish [initial body weight (51.18±1.14)g] as exprimental object were randomly divided into 6 groups with 3 replicates per group and 40 individuals per replicate. The results showed that: ① Compared with the control group S, the addition of kelp enzymatic extract in diet had no significant effect on the suvival rate(SR) of channel catfish, the specific growth rate(SGR) increased by 1.20% to 5.39%, and the feed coefficient rate(FCR) decreased by −0.83% to 9.09%. A quadratic polynomial regression analysis was carried out with SGR and FCR as the targets respectively, and the optimal addition amount of kelp enzymatic hydrolysis extract in the feed was 0.98 and 0.96 g/kg. The condition factor(CF) and viscero-somatic index(VSI) decreased significantly in groups KP10 to KP20, and the hepatosomatic index(HSI) decreased significantly in group KP10. The moisture, crude fat, crude protein and ash of fish had no significantly difference in all groups. ② Height of duplicature in group KP3, height and width of duplicature and thickness of muscularis in group KP5, thickness of muscularis in group KP10 were all significantly higher than those in group S. ③ Total RNA was extracted from S and KP10 groups’ hepatopancreas. Transcriptome sequencing results showed that there were 81 significantly down-regulated genes and 199 up-regulated genes. GO functional classification analysis results indicated that differentially expressed genes(DEGs) were annotated to items such as DNA transcription, metal ion binding, membrane and membrane composition. According to the results of KEGG pathway analysis, we found that DEGs were mainly enriched in 12 related pathways including cell proliferation and differentiation, hormone regulation, lipid metabolism, carbohydrate metabolism, growth factor metabolism and so on. Our studies have indicated that adding kelp enzymatic extract to diet can promote the growth of I. punctatus, reduce feed coefficient, and have positive effects on the intestinal morphology and the glucose and lipid metabolic capability of liver and pancreas. The suitable addition amount was 0.96-0.98 g/kg.
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Effects of Saccharina japonica enzymatic hydrolysis extract on the growth and hepatopancreas transcriptome of Ictalurus punctatus

    Corresponding author: YE Yuantu, yeyt@suda.edu.cn
  • 1. Key Laboratory of Aquatic Animal Nutrition of Jiangsu Province, School of Basic Medicine and Biological Sciences, Soochow University, Suzhou 215123, China
  • 2. Shandong Rongcheng Hongde Marine Biological Technology Co., Ltd, Rongcheng    264300, China
  • 3. Wuxi Sanzhi Biotechnology Co., Ltd., Wuxi    214000, China

Abstract: The aim of this study was to investigate the effects of kelp enzymatic extract on the growth and hepatopancreas transcriptome of Ictalurus punctatus. We prepared different diets with enzymatic extract of kelp addition amount of 0 g/kg (S), 0.3 g/kg (KP3), 0.5 g/kg (KP5), 1.0 g/kg (KP10), 1.5 g/kg (KP15), 2.0 g/kg (KP20) to feed channel catfish in cage-culture for 60 days. Channel catfish [initial body weight (51.18±1.14)g] as exprimental object were randomly divided into 6 groups with 3 replicates per group and 40 individuals per replicate. The results showed that: ① Compared with the control group S, the addition of kelp enzymatic extract in diet had no significant effect on the suvival rate(SR) of channel catfish, the specific growth rate(SGR) increased by 1.20% to 5.39%, and the feed coefficient rate(FCR) decreased by −0.83% to 9.09%. A quadratic polynomial regression analysis was carried out with SGR and FCR as the targets respectively, and the optimal addition amount of kelp enzymatic hydrolysis extract in the feed was 0.98 and 0.96 g/kg. The condition factor(CF) and viscero-somatic index(VSI) decreased significantly in groups KP10 to KP20, and the hepatosomatic index(HSI) decreased significantly in group KP10. The moisture, crude fat, crude protein and ash of fish had no significantly difference in all groups. ② Height of duplicature in group KP3, height and width of duplicature and thickness of muscularis in group KP5, thickness of muscularis in group KP10 were all significantly higher than those in group S. ③ Total RNA was extracted from S and KP10 groups’ hepatopancreas. Transcriptome sequencing results showed that there were 81 significantly down-regulated genes and 199 up-regulated genes. GO functional classification analysis results indicated that differentially expressed genes(DEGs) were annotated to items such as DNA transcription, metal ion binding, membrane and membrane composition. According to the results of KEGG pathway analysis, we found that DEGs were mainly enriched in 12 related pathways including cell proliferation and differentiation, hormone regulation, lipid metabolism, carbohydrate metabolism, growth factor metabolism and so on. Our studies have indicated that adding kelp enzymatic extract to diet can promote the growth of I. punctatus, reduce feed coefficient, and have positive effects on the intestinal morphology and the glucose and lipid metabolic capability of liver and pancreas. The suitable addition amount was 0.96-0.98 g/kg.

  • 斑点叉尾鮰(Ictalurus punctatus)自1984被引进后经过三十多年的推广,被广大养殖户接受并广泛养殖,是我国重要的淡水养殖名优品种[1]。随着养殖规模扩大,饲料用量不断增加,饲料资源的匮乏将成为巨大的挑战。通过鉴定新饲料或开发提高资源利用效率的新添加剂来扩大饲料资源基础,将对斑点叉尾鮰养殖的可持续发展发挥重要作用[2]

    海带(Saccharina japonica)是一种冷温带性大型底栖藻类,主要分布于北太平洋和大西洋沿海。我国海带的养殖面积和产量均居世界首位,到2018年我国海带的年产量已超过150万t[3]。除了资源丰富、价格低廉等特点,海带还具有较高的营养价值,包括丰富的碳水化合物(褐藻胶、海带淀粉、褐藻糖胶、甘露醇)、不饱和脂肪酸、矿物质和维生素等营养成分[4]。其中,褐藻胶具有多种生物活性,并且可以作为一种有效的饲料赋形剂[5]。褐藻糖胶和海带淀粉这2种海带多糖均具有清除自由基的能力和抗氧化作用[6],可以增强机体免疫[7]。海带兼具营养和免疫的双重功效,具有作为动物饲料添加剂的巨大潜力[8]。已有不少报道表明,凡纳滨对虾(Litopenaeus vannamei)[9]、黑鲍[Haliotis discus hannai (♀)×H. discus discus (♂)][10]、异育银鲫(Carassius auratus gibelio)[11]等水产动物摄食添加海带的饲料后,生长性能均有不同程度的提高。然而,海带细胞壁中存在大量的褐藻胶,其水溶性很差,阻碍了组织间的营养因子和活性物质溶出,海带饲料的应用也因此受限[12]。不同的加工工艺可以改善这一局限性,例如热提取法、酸碱提取法、酶解提取法、超声波提取法和发酵等[13],这些方法可以提高海带营养物质的利用效率。其中酶解法具有水解位点专一、高效、反应条件温和等优势,符合实际生产需求。本实验使用的海带酶解提取物是应用酶解技术将纤维质和果胶质水解,提取出细胞质和细胞间质液中的物质,再经分离纯化定向浓缩获得的产品。海带酶解提取物对比海带粉,在饲料中的添加量更低、溶解性更好,有利于消化吸收,能更充分发挥海带的营养价值。

    本实验以斑点叉尾鮰为对象,在日粮中添加不同比例的海带酶解提取物,研究其对斑点叉尾鮰生长、肠道组织结构的影响,通过肝脏转录组基因差异表达结果分析肝脏的代谢状态,为海带酶解提取物作为斑点叉尾鮰饲料添加剂的使用效果和适宜添加量提供参考。

1.   材料与方法
  • 斑点叉尾鮰购自大丰市华辰水产科技有限公司养殖基地,鱼种初始体质量为(51.18±1.14) g,驯养1周后,选用规格一致、体质健康的鱼种随机分成6组,每组3个重复,共18个养殖单元(网箱),每个单元40尾,共720尾。

  • 饲料中添加的海带酶解提取物购自山东荣成海洋生物科技有限公司,海带酶解提取物的组成成分见表1。以不添加海带酶解提取物的饲料组为对照组(S)。海带酶解提取物在饲料中的添加量分别为0.3 g/kg (KP3)、0.5 g/kg (KP5)、1.0 g/kg (KP10)、1.5 g/kg (KP15)、2.0 g/kg (KP20)。饲料组成及营养水平见表2。饲料原料粉碎后过60目筛,混匀加适量水搅拌,使用华祥牌HKj200制粒机加工成直径为1.5 mm,长度为4 mm左右的颗粒,风干后置于4 °C保存待用。

    海藻酸
    alginate
    海带多糖
    kelp polysaccharide
    海带寡糖
    kelp oligosaccharides
    蛋白质
    protein
    甘露醇
    mannitol

    potassium

    calcium

    iodine
    28.279.1017.703.5014.2618.600.770.05

    Table 1.  Composition of S. japonica enzymatic hydrolysis extract %

    成分
    component
    SKP3KP5KP10KP15KP20
    原料 ingredients
    面粉 flour 10.00 10.00 10.00 10.00 10.00 10.00
    米糠 rice bran 5.00 5.00 5.00 5.00 5.00 5.00
    米糠粕 rice bran meal 7.00 6.97 6.95 6.90 6.85 6.80
    膨化大豆 expanded soybean 8.00 8.00 8.00 8.00 8.00 8.00
    豆粕 soybean meal 26.80 26.80 26.80 26.80 26.80 26.80
    菜粕 rapeseed meal 14.00 14.00 14.00 14.00 14.00 14.00
    进口鱼粉65 imported fish meal 65 12.00 12.00 12.00 12.00 12.00 12.00
    美国鸡肉粉 American chicken meal 6.00 6.00 6.00 6.00 6.00 6.00
    磷酸二氢钙 Ca(H2PO4)2 2.20 2.20 2.20 2.20 2.20 2.20
    沸石粉 zeolite meal 2.00 2.00 2.00 2.00 2.00 2.00
    膨润土 bentonite 2.00 2.00 2.00 2.00 2.00 2.00
    豆油 soybean oil 4.00 4.00 4.00 4.00 4.00 4.00
    海带酶解提取物 kelp enzymatic hydrolysis extract 0.00 0.03 0.05 0.10 0.15 0.20
    预混料 premix feed1 1.00 1.00 1.00 1.00 1.00 1.00
    合计 total 100.00 100.00 100.00 100.00 100.00 100.00
    营养水平 nutrient levels
    水分 moisture 6.63 6.74 6.02 5.88 5.72 6.06
    粗蛋白质 crude protein 36.01 35.92 36.22 36.82 37.21 37.26
    粗脂肪 crude lipid 9.42 9.38 9.49 9.52 9.58 9.53
    粗灰分 crude ash 12.10 12.14 12.17 12.20 12.30 12.26
    磷 phosphorus 1.44 1.51 1.48 1.43 1.50 1.51
    注:1.每kg预混料含铜 2.5 mg,铁 64 mg,锰 13 mg,锌 19 mg,碘 0.021 mg,硒 0.07 mg,钴 0.016 mg,镁 96 mg,钾 0.05 mg,维生素A 0.8 mg,维生素B1 0.8 mg,维生素B2 0.8 mg,维生素B6 1.2 mg,维生素B12 0.002 mg,维生素C 30 mg,泛酸钙 2.5 mg,烟酸 2.5 mg,维生素D3 0.3 mg,维生素K3 0.5 mg,叶酸 0.5 mg,肌醇 10 mg
    Notes: 1. one kilogram of premix contain Cu 2.5 mg, Fe 64 mg, Mn 13 mg, Zn 19 mg, I 0.021 mg, Se 0.07 mg, Co 0.016 mg, Mg 96 mg, K 0.05 mg, VA 0.8 mg, VB1 0.8 mg, VB2 0.8 mg, VB6 1.2 mg, VB12 0.002 mg, VC 30 mg, calcium pantothenate 2.5 mg, niacin 2.5 mg, VD3 0.3 mg, VK3 0.5 mg, folic acid 0.5 mg, inositol 10 mg

    Table 2.  Composition and nutrient levels of experimental diets (AD basis) %

  • 养殖实验在盐城市大丰区华辰水产实业有限公司养殖基地池塘的实验网箱中进行,网箱规格为1.5 m × 1.8 m × 1.8 m。池塘中设置1.5 kW的叶轮式增氧机1台,运行7 h/d。日投喂2次(07:00、16:00),日投喂量为鱼体质量的3%~4%,每10 d估算1次鱼体质量并调整投喂量。正式实验从2018年7月8日—9月8日,共计60 d,养殖期间水温24.0 ~32.5 °C。实验期间溶解氧含量>7.0 mg/L,pH 7.8~8.2,氨氮浓度<0.10 mg/L,亚硝酸盐浓度<0.005 mg/L,硫化物浓度<0.05 mg/L。

  • 养殖实验结束禁食24 h后进行采样。记录实验鱼尾数并称重,用于生长速率、饲料系数的计算。取10尾鱼测量体长、体质量,编号后解剖,称取内脏团总质量以及肝脏质量,用于形体指标计算。

  • 每个网箱随机取2尾鱼,每组保留6尾鱼全鱼样品,进行常规体成分测定。饲料原料及所有实验鱼用粉碎机粉碎均匀,在LGJ-18B型冷冻干燥机中干燥至恒重测定水分;采用凯式定氮法(GB 5009.5—2010)测定粗蛋白质含量;采用索氏抽提法(GB/T 14772—2008)测定粗脂肪含量;采用灼烧法 (GB 5009.4—2010) 测定灰分含量;采用比色法 (GB 5009.87—2016) 测定总磷含量。

  • 每个网箱取2尾鱼,每组6尾鱼,常规解剖分离出中肠 (肠道全长的1/2处),取中肠10 mm肠段,甲醛固定,按常规组织切片法进行脱水、透明、透蜡、石蜡包埋后切片,切片厚度5~6 μm,H.E染色,切片在光学显微镜下观察记录微观形态结构特征,显微测量,并拍照留存。

    皱襞高度用黏膜皱襞顶端至基部凹陷处的垂直距离表示,每张切片随机选取10个皱襞测量,取平均值作为结果;皱襞宽度以皱襞基部、高度1/3和2/3处的平均宽度表示,每张切片随机选取10个皱襞测量,取平均值作为结果;每张切片随机选取10处肌层厚度进行测量,取平均值作为结果。

  • 每个网箱取3尾鱼,每组9尾鱼,用MS-222麻醉后解剖出肝脏组织,将每个网箱3尾鱼体的肝脏等量混合后(每个组3个平行样)放入1.5 mL EP管中,液氮速冻运回实验室−80 °C保存备用。将生长结果梳理后,选取特定生长率最高的实验组(KP10)和对照组的肝脏组织进行转录组测序。

  • 将肝脏组织置于研体中加入液氮磨至粉末,用TRIzol (Invitrogen, Carlsbad, CA, USA)方法对总样品的RNA进行分离和纯化。然后用NanoDrop ND-1000 (NanoDrop, Wilmington, DE, USA)对总RNA的量与纯度进行质控。再通过Agilent 2100对RNA的完整性进行检测,以RIN number>7.0为合格的标准。

  • cDNA文库的制备及测序委托杭州联川生物技术股份有限公司完成,具体方法:取出5 μg的质检合格的总RNA,使用oligo(dT)磁珠,通过两轮的纯化对其中的带有PolyA(多聚腺苷酸)的mRNA进行特异性捕获。将捕获到的mRNA进行片段化,再通过逆转录酶的作用合成cDNA。然后使用E. coli DNA polymerase I与RNase H进行二链合成,将这些DNA与RNA的复合双链转化成DNA双链,同时掺入DUTP,补齐平末端。再在其两端各加上一个A碱基,使其与末端带有T碱基的接头进行连接,利用磁珠进行筛选和纯化。以UDG酶消化二链,再通过PCR,使其形成片段大小为300 bp (±50 bp)的文库。最后,使用Illumina Hiseq 4000 (LC Bio, China)对其进行双端测序,读长150 bp。

  • 依据基因在样本中的表达量,先计算表达量的倍数,再计算以2为底、差异表达倍数(fold change)的对数值,即log2 (FC)值。log2 (FC)值为正表示差异表达上调,为负表示差异表达下调。以|log2 (FC)|≥1同时P<0.05作为显著差异的阈值,筛选出达到该条件的基因为显著差异表达基因。然后对差异表达基因进行GO富集性分析和KEGG信号通路富集性分析。

  • 原始数据经Excel 2010软件初步整理后,用SPSS 18.0软件进行单因素方差分析(One-Way ANOVA),同时进行Duncan氏多重比较分析实验数据的差异显著性,结果以平均值±标准差(mean±SD)表示,以P<0.05为差异显著水平。

2.   结果
  • 添加海带酶解提取物对各组斑点叉尾鮰存活率均无显著影响(P>0.05)。各组的特定生长率均高于S组,其中KP5和KP10组与S组相比,特定生长率分别提高了4.19%和5.39% (P<0.05);除KP20组外各组的饲料系数均低于S组,KP10组降低了9.09% (P<0.05)。结果显示日粮添加海带酶解提取物可以提高斑点叉尾鮰生长速率、降低饲料系数(表3)。日粮添加低剂量海带酶解提取物后斑点叉尾鮰的脏体比、肝体比与对照组相比均有不同程度降低,KP10、KP15、KP20组肥满度和脏体比显著降低(P<0.05),KP10组肝体比显著降低(P<0.05)。各组斑点叉尾鮰鱼体的水分、粗脂肪、粗蛋白质和粗灰分含量无显著变化(P>0.05)(表4)。

    项目
    items
    SKP3KP5KP10KP15KP20
    初始体质量/g IBW 52.34±0.83 50.26±1.13 50.75±0.69 51.52±1.11 51.43±1.06 50.84±1.70
    终末体质量/g FBW 144.48±1.66bc 141.57±3.91abc 144.39±4.84bc 148.26±3.16c 140.62±0.71abc 141.33±6.51abc
    存活率/% SR 96.67±3.82 95.83±3.82 99.17±1.44 100 100 100
    特定生长率/(%/d) SGR 1.67±0.01a 1.73±0.03b 1.74±0.08b 1.76±0.02b 1.69±0.01ab 1.70±0.03ab
    与S组比较/% compared with group S 3.59 4.19 5.39 1.20 1.80
    饲料系数 FCR 1.21±0.05b 1.19±0.02b 1.15±0.06ab 1.10±0.03a 1.19±0.01b 1.22±0.03b
    与S组比较/% compared with group S −1.65 −4.96 −9.09 −1.65 0.83
    注:同行肩标不同小写字母表示差异显著(P<0.05),下同
    Notes:values with different lowercase superscripts mean significant difference(P<0.05), the same below

    Table 3.  Effects of S. japonica enzymatic hydrolysis extract added in feed on growth performance and FCR of I. punctatus n=3

    项目
    items
    SKP3KP5KP10KP15KP20
    形体指标 body parameters (n=10)
    肥满度/(g/cm3) CF 1.53±0.06bc 1.57±0.02c 1.48±0.05b 1.39±0.02a 1.41±0.03a 1.38±0.03a
    脏体比/% HSI 8.18±0.95c 8.09±1.06bc 7.76±0.69bc 5.89±0.67a 6.71±1.17ab 6.69±0.49ab
    肝体比/% VSI 1.16±0.13b 1.09±0.14ab 1.07±0.12ab 0.96±0.09a 1.08±0.04ab 1.07±0.04ab
    体成分 body composition (n=3)
    水分/% moisture 65.06±1.46 67.54±1.58 65.73±1.86 68.89±1.75 67.01±1.58 69.30±0.83
    粗蛋白质/% crude protein 16.32±0.25 14.47±0.97 16.21±0.57 15.29±0.33 15.74±0.61 14.23±0.90
    粗脂肪/% crude liqid 14.44±1.39 12.89±1.51 13.56±1.22 11.37±2.20 12.22±2.26 11.96±0.75
    粗灰分/% crude ash 3.86±0.84 3.46±0.07 3.53±0.48 3.47±0.41 4.00±0.53 3.56±0.23

    Table 4.  Effects of S. japonica enzymatic hydrolysis extract on the body parameters and body composition of I. punctatus

    随着饲料中海带酶解提取物添加量的增加,斑点叉尾鮰的特定生长率呈现二次回归方程的变化趋势,表明海带酶解提取物在日粮中的添加量有剂量效应特征。特定生长率与海带酶解提取物添加量之间符合一元二次回归方程y=−0.060 2x2+0.118 5x+1.686 4 (R2=0.552 5),经计算得出当以生长速率为标准时,海带酶解提取物在饲料中的最适添加量为0.98 g/kg (图1)。饲料中海带酶解提取物添加量与饲料系数的关系符合一元二次回归方程y=0.091 2x2−0.175 5x+1.2163 (R2=0.747 8),经计算得出当以饲料系数为标准时,海带酶解提取物在饲料中的最适添加量为0.96 g/kg(图2)。

    Figure 1.  Relationship between specific growth rate of I. punctatus and the addition amount of S. japonica enzymatic hydrolysis extract

    Figure 2.  Relationship between feed conversion ratio of I. punctatus and the addition amount of S. japonica enzymatic hydrolysis extract

  • 斑点叉尾鮰养殖60 d后,取中肠做组织切片,进行光学显微镜观察。

    肠道组织切片观察结果显示,各组肠道皱襞高度、皱襞宽度和肌层厚度的量化结果显示,随着海带酶解提取物添加量增加,各组鱼肠道皱襞高度和宽度都呈现先增加后减少的趋势(图版图3)。与对照组相比,KP3、KP5组皱襞高度分别增加了11.98%和19.29% (P<0.05);KP5、KP10组肠道皱襞宽度增加了46.10%和39.76% (P<0.05);KP5组肠道肌层厚度增加了26.10% (P<0.05)。结果显示,日粮添加0.3~1.0 g/kg海带酶解提取物在一定程度上改善了斑点叉尾鮰的肠道结构,而更高的添加量没有明显改善效果。

    Figure 图版.  Effect of S. japonica enzymatic hydrolysis extract on the intestinal structure of I. punctatus (H.E staining, 200×)

    Figure 3.  Effects of S. japonica enzymatic hydrolysis extract on intestinal structure of I. punctatus

  • 将对照组与添加海带酶解提取物组肝脏转录组数据进行统计分析,差异表达显著下调的基因数为81个,上调的基因数为199个,差异表达显著上调的基因数远大于下调基因数(表5)。

    log2(FC)下调基因数
    down-regulated gene number
    log2(FC)上调基因数
    up-regulated gene number
    ≤(−1)~(−2) 72 ≥1~2 142
    ≤(−2)~(−4) 8 ≥2~4 55
    ≤(−4) 1 ≥4 2
    小计 subtotal 81 199
    差异表达基因总数
    total number of differentially expressed genes
    7 464 12 264

    Table 5.  Statistics on the differentially expressed genes of transcriptome between common diet and diet supplemented with S. japonica enzymatic hydrolysis extract

    筛选出海带酶解提取物相比对照组上调或下调幅度最大的前10位基因,其GO注释和KEGG通路的注释结果显示:显著上调的基因中有3个已知基因,分别为核受体共激活因子2(ncoa2)、Krüppel样因子12亚型 (klf12)和包含BTB结构域的锌指蛋白16 (zbtb16);显著下调的基因有2个已知基因,分别为α-1肾上腺素能受体(adra1b)和胶原蛋白α-1 (24)链 (col24a1) (表6)。

    基因
    gene
    log2(FC)基因描述
    gene description
    GO注释
    GO annotations
    KEGG通路
    KEGG pathway
    P
    P value
    LOC108268537 5.43 未鉴定的蛋白质
    uncharacterized protein
    GO:0070989氧化脱甲基
    oxidative demethylation
    NA 3.39×10−12
    ncoa2 2.86 核受体共激活因子2
    nuclear receptor coactivator 2
    GO:0003713转录共激活子活性
    transcription coactivator
    NA 2.89×10−5
    LOC108271496 2.52 1,25-二羟维生素D3 24-羟化酶,线粒体
    1,25-dihydroxyvitamin D3 24-hydroxylase, mitochondrial
    GO:0016491氧化还原酶活性
    oxidoreductase activity
    00100 类固醇生物合成
    steroid biosynthesis
    1.40×10−4
    c5h1orf194 2.48 未表征蛋白C1orf194同源物
    uncharacterized protein C1orf194 homolog
    NA NA 2.29×10−4
    LOC108271131 2.30 脂蛋白脂肪酶样前体
    lipoprotein lipase-like precursor
    GO:0004465脂蛋白脂肪酶活性
    lipoprotein lipase activity
    03320 PPAR信号通路
    PPAR signaling pathway
    4.09×10−4
    klf12 2.22 Krüppel样因子12亚型 X1
    Krueppel-like factor 12 isoform X1
    GO:0046872金属离子结合
    metal ion binding
    NA 7.78×10−4
    LOC108267282 2.14 嘌呤核苷磷酸化酶样
    purine nucleoside phosphorylase-like
    GO:0004731嘌呤核苷磷酸化酶活性
    purine-nucleoside phosphorylase activity
    00760 烟酸酯和烟酰胺代谢
    nicotinate and nicotinamide metabolism
    9.17×10−4
    zbtb16 2.12 包含BTB结构域的锌指蛋白16
    zinc finger and BTB domain-containing protein 16
    GO:0006355转录调控,DNA模板
    regulation of transcription, DNA-templated
    NA 1.07×10−3
    LOC108271807 2.06 维生素D3羟化酶相关蛋白样亚型X1
    vitamin D3 hydroxylase-associated protein-like isoform X1
    GO:0016021膜的整体成分
    integral component of membrane
    04723逆行内源性大麻素信号
    retrograde endocannabinoid signaling
    1.56×10−3
    LOC108270518 1.97 C-C基序趋化因子样21aC-C
    motif chemokine 21a-like
    GO:0002548单核细胞趋化性
    monocyte chemotaxis
    04672产生IgA的肠道免疫网络
    intestinal immune network for IgA production
    2.24×10−3
    LOC108258483 −3.12 ras相关和血小板同源结构域包含蛋白1样亚型
    ras-associated and pleckstrin homology domains-containing protein 1-like isoform X1
    GO:0007165信号转导
    signal transduction
    04611 血小板活化
    platelet activation
    2.18×10−5
    LOC100304637 −3.19 胰岛素样生长因子2
    IGF2
    GO:0005159胰岛素样生长因子受体结合
    insulin-like growth factor receptor binding
    04010 MAPK信号通路
    MAPK signaling pathway
    1.67×10−5
    LOC108259607 −3.26 未鉴定的蛋白质
    uncharacterized protein
    GO:0006412翻译
    translation
    04146 过氧化物酶体
    peroxisome
    1.84×10−5
    LOC108255161 −3.29 核受体亚家族1 D组成员2样
    nuclear receptor subfamily 1 group D member 2-like
    NA NA 9.46×10−6
    adra1b −3.33 α-1肾上腺素能受体
    alpha-1B adrenergic receptor
    GO:0001994去甲肾上腺素-肾上腺素血管收缩参与全身动脉血压调节
    norepinephrine-epinephrine vasoconstriction involved in regulation of systemic arterial blood pressure
    04080 神经活性配体-受体
    neuroactive ligand-receptor interaction
    1.16×10−5
    LOC108267421 −3.38 视网膜鸟苷基环化酶2样Ix2亚型
    retinal guanylyl cyclase 2-like isoform X1
    GO:0000166核苷酸结合
    nucleotide binding
    00230嘌呤代谢
    purine metabolism
    6.67×10−6
    LOC108259372 −3.50 转导蛋白样增强蛋白3-B同工型X4
    transducin-like enhancer protein 3-B isoform X4
    GO:0030099骨髓细胞分化
    myeloid cell differentiation
    04330 Notch信号通路
    Notch signaling pathway
    3.88×10−6
    col24a1 −3.96 胶原蛋白α-1 (24) 链
    collagen alpha-1(ⅩⅩⅣ) chain
    GO:0005201细胞外基质结构成分
    extracellular matrix structural constituent
    NA 2.00×10−7
    LOC108255274 −4.59 核受体亚家族4 A组成员1样
    nuclear receptor subfamily 4 group A member 1-like
    GO:0003700DNA结合转录因子活性
    DNA binding transcription factor activity
    04010 MAPK信号通路
    MAPK signaling pathway
    4.55×10−9
    LOC108254794 −5.64 25-羟基维生素D-1α羟化酶,线粒体
    25-hydroxyvitamin D-1 alpha hydroxylase, mitochondrial
    GO:0004498钙糖二醇1-单加氧酶活性
    calcidiol 1-monooxygenase activity
    00100 甾体生物合成
    steroid biosynthesis
    3.54×10−12
    注:NA. 未获得
    Notes: NA. not available

    Table 6.  Top 10 genes with the greatest up/down-regulation in the S. japonica enzymatic hydrolysis extract group compared with the control group

  • 将经过GO注释的显著差异表达基因进行分类,在生物过程条目中,DNA转录、信号转导相关基因受影响较大,且均表现为差异表达上调。在分子功能条目中,受影响较大的为结合类,包括金属离子结合、ATP结合、DNA结合、核苷酸结合、核酸结合、锌离子结合等基因,相关基因以差异表达上调为主;其次为催化活性,包括转移酶、水解酶、蛋白激酶活性,其差异表达显著上调基因数分别占显著差异基因数的65%、60%和58.3%。在细胞组成条目中,膜及膜组成成分的相关差异表达显著基因数最多,分别为57和51个,核、细胞质、细胞外区域也分别有49、41和12个显著差异表达的相关基因(表7)。这些结果表明摄食添加海带酶解提取物的日粮对鱼体细胞组成、细胞信号转导和分子功能方面产生了重大影响,从而可能导致细胞组织结构和功能发生改变。富集的GO Term大部分与细胞信号转导相关,而细胞信号转导系统具有调节细胞增殖、分化、代谢、应激、防御和凋亡等作用,从显著差异上调与下调的基因比例,多数差异基因表达上调,因此添加海带酶解提取物可能对促进细胞增殖、分化以及加强代谢、免疫防御等方面有积极作用。

    GO 功能
    GO function
    GO ID
    gene ontology ID
    GO信息
    GO Term
    显著差异表达基因数
    number of genes with
    significant differences
    上调基因数
    up-regulated
    gene number
    下调基因数
    down-regulated
    gene number
    生物过程
    biological process
    GO:0006355 以DNA为模板的转录调控
    regulation of transcription, DNA-templated
    25 15 10
    GO:0006351 转录,DNA模板
    transcription, DNA templated
    19 8 11
    GO:0008150 生物过程
    biological process
    17 12 5
    GO:0006468 蛋白质磷酸化
    protein phosphorylation
    15 10 5
    GO:0035556 细胞内信号转导
    intracellular signal transduction
    14 12 2
    GO:0007165 信号转导
    signal transduction
    13 12 1
    GO:0055114 氧化还原过程
    oxidation-reduction process
    12 11 1
    分子功能
    molecular function
    GO:0046872 金属离子结合
    metal ion binding
    46 38 8
    GO:0005524 ATP结合
    ATP binding
    25 15 10
    GO:0003677 DNA结合
    DNA binding
    23 16 7
    GO:0003674 分子功能
    molecular function
    20 14 6
    GO:0016740 转移酶活性
    transferase activity
    20 13 7
    GO:0000166 核苷酸结合
    nucleotide binding
    16 7 9
    GO:0016787 水解酶活性
    hydrolase activity
    15 9 6
    GO:0003676 核酸结合
    nucleic acid binding
    14 8 6
    GO:0003700 DNA结合转录因子活性
    DNA binding transcription factor activity
    14 10 4
    GO:0004672 蛋白激酶活性
    protein kinase activity
    12 7 5
    GO:0008270 锌离子结合
    zinc ion binding
    10 8 2
    细胞组分
    cellular component
    GO:0016020
    membrane
    57 42 15
    GO:0016021 膜的组成部分
    integral component of membrane
    51 37 14
    GO:0005634
    nucleus
    49 31 18
    GO:0005737 细胞质
    cytoplasm
    41 27 14
    GO:0005575 细胞成分
    cellular component
    27 20 7
    GO:0005886 质膜
    plasma membrane
    14 10 4
    GO:0005576 细胞外区域
    extracellular region
    12 9 3

    Table 7.  GO Term classification results of differentially expressed genes in liver transcriptome after ingestion of diet with S. japonica enzymatic hydrolysis extract

  • 在生物体内,不同基因相互协调行使其生物学功能,基于KEGG Pathway的分析有助于更进一步解读基因的生物学功能。KEGG通路分类结果显示,差异基因显著富集在脂肪细胞因子信号通路、FoxO信号通路、类固醇生物合成通路等共12个代谢通路上(P<0.05)(表8)。这些信号通路主要包括细胞增殖与分化(FoxO信号通路、铁死亡、P53信号通路、TGF-beta信号通路)、激素调控(胰岛素信号通路)、脂类代谢(脂肪细胞因子信号通路、类固醇生物合成)、糖类代谢(半乳糖代谢、淀粉和蔗糖代谢、糖酵解/糖异生)以及生长因子代谢(磷酸肌醇代谢、烟酸酯和烟酰胺代谢)等方面,同时由于差异表达上调基因数大于差异表达下调的基因数,因此多为对信号通路产生正面影响,尤其是对细胞增殖和营养因子代谢有促进作用。最终结果表现为鱼体增强对营养物质的吸收和利用,鱼体生长速率提高。

    通路ID
    pathway ID
    通路
    pathway
    显著差异表达基因数
    number of genes with
    significant differences
    上调基因数
    up-regulated
    gene number
    下调基因数
    down-regulated
    gene number
    ko04920 脂肪细胞因子信号通路 adipocytokine signaling pathway 8 6 2
    ko04068 FoxO信号通路 FoxO signaling pathway 9 6 3
    ko00100 类固醇生物合成 steroid biosynthesis 3 2 1
    ko04216 铁死亡 ferroptosis 4 3 1
    ko04115 P53信号通路 P53 signaling pathway 5 4 1
    ko00562 磷酸肌醇代谢 inositol phosphate metabolism 6 5 1
    ko00052 半乳糖代谢 galactose metabolism 3 1 2
    ko00760 烟酸酯和烟酰胺代谢 nicotinate and nicotinamide metabolism 3 2 1
    ko00500 淀粉和蔗糖代谢 starch and sucrose metabolism 3 1 2
    ko04910 胰岛素信号通路 insulin signaling pathway 7 5 2
    ko04350 TGF-beta信号通路 TGF-beta signaling pathway 5 4 1
    ko00010 糖酵解/糖异生 glycolysis/gluconeogenesis 4 2 2

    Table 8.  KEGG pathway classification results of differentially expressed genes in the liver transcriptome after ingestion of diets supplemented with S. japonica enzymatic hydrolysis extract

3.   讨论
  • 目前已有较多文献报道海藻在饲料添加剂方面的应用,然而不同海藻在不同种类鱼体内的最适添加量差异显著,不同海藻在同一种鱼类体内的最适添加量也有不同。为了更准确反映适宜添加量,本实验设置了海带酶解提取物5个不同添加梯度,结果显示,日粮添加0.3~2.0 g/kg的海带酶解提取物均在一定程度上提高了斑点叉尾鮰的特定生长率,添加0.3~1.5 g/kg的海带酶解提取物可以降低饲料系数。当添加量为0.98 g/kg时对斑点叉尾鮰的生长促进效果最好。

    实验用的海带酶解提取物主要成分是褐藻酸钾以及海带多糖和寡糖。褐藻酸钾可以与饲料中的阳离子反应形成交联键,生成凝胶体将饲料原料包裹固定。这种凝胶体轻微吸水膨胀,能保持长时间不溃散,提高了饲料的黏结性、耐水性和适口性,从而提高饲料的利用效率[12]。有研究表明,将2%~5%的海藻作为黏结剂添加到肉鸡饲料中,其饲料的颗粒稳定性指数(pellet durability index,PDI)和硬度有所提高,且肉鸡的饲料效率也显著提高[13]

    对于海带酶解提取物的促生长作用机制已有较多报道,其作用机制是多方面的。首先由于藻类中还普遍存在一些如二甲基-β-丙酸噻亭(DMPT)和二甲基巯基丙酸(DMSP)等具有诱食作用的物质[14-15],通过这些成分刺激鱼类的化学感觉器官,进而刺激中枢神经释放促摄食因子,提高鱼类采食量。目前的大部分研究主要集中在营养水平,与海带酶解提取物的诱食作用相关的研究较少,其发挥作用的成分和应用效果有待进一步研究。海带的营养价值众所周知,除了褐藻胶、甘露醇等碳水化合物外,海带中尤其富含碘、矿物质、维生素、色素以及其他活性成分,饲料中添加海带或其酶解提取物可以有效改善饲料营养素组成和饲料性质,这也是将海带产品作为饲料添加剂的主要原因。有研究发现海带酶解提取物的多糖成分可以通过提高消化酶活性来促进鱼体对营养物质的吸收。李文武[16]的实验结果表明,海带多糖能显著提高斜带石斑鱼(Epinephelus coioides)胃肠蛋白酶活性和肠黏膜麦芽糖酶活性,但对乳糖和蔗糖酶影响较弱。杨晴等[17]的研究也发现,褐藻糖胶对黄颡鱼(Pelteobagrus fulvidraco)幼鱼胃和肠道脂肪酶活性具有增强作用。此外还有研究证明,褐藻糖胶对人体和动物消化道菌群结构有一定的影响。肠道内的乳酸杆菌(Lactobacillus)、双歧杆菌(Bifidobacterium)等优势菌群是重要的生理性细菌,在调节肠道微生态平衡,抑制病原菌生长和腐败菌发育,调控宿主的营养代谢、生理机能、免疫机能等方面有着重要的影响[18]。0.04%的褐藻寡糖可以提高盲肠乳酸杆菌的数量,0.2%的褐藻酸钠和褐藻寡糖明显降低大肠杆菌(Escherichia coli)的数量[19]。海带通过促进优势菌群的增殖以及抑制病原菌生长来维持肠道菌群的平衡从而促进生长[20]。与众多研究结果相似的是,本实验在日粮中添加较高剂量的海带酶解提取物对斑点叉尾鮰的促生长作用反而有所降低,表现出剂量效应,即存在最适添加量的问题。一方面是因为海带酶解提取物中大量的海带多糖往往会对营养物质的吸收产生阻碍。本实验转录组结果表现出的是海带酶解提取物中含有某些成分可能对促进脂质代谢有积极作用,然而当剂量达到一定程度,最终会表现为减少脂肪的利用。其主要成分褐藻胶本身对胆固醇、胆汁酸具有很强的吸附性,同时作为酸性多糖将带有大量负电荷,进一步阻止胆固醇进入细胞,通过吸附结合胆汁酸、胆固醇降低了鱼体对胆汁酸、胆固醇的重吸收[21]。此外,海带多糖作为一种潜在的膳食纤维,具有促进肠道蠕动的作用,但是加快肠道转运的同时也会减少食物在肠道的停留时间,斑点叉尾鮰作为一种偏肉食性的鱼类,肠道长度与草食性鱼类相比偏短,这可能会导致营养物质来不及充分吸收便排出体外,从而降低了其生长性能。

  • 肠道是鱼体与饲料直接接触的重要器官,其皱襞密度、高度、厚度以及肌层的厚度会直接影响营养物质的吸收[22]。本实验中,日粮添加0.3~1.0 g/kg海带酶解提取物可以增加皱襞高度、宽度和肠壁肌层厚度,从而增大肠道与营养物质的接触面积并且增强肠道蠕动的动力,表明较低剂量海带酶解提取物可以改善肠道组织形态结构,这可能归因于海带多糖中的褐藻糖胶对大肠杆菌、双歧杆菌的抑制作用[20]。同时褐藻糖胶也可以抑制肠球菌以及链球菌等菌种在肠道的附着[23],避免了肠道组织形态被病原菌破坏,健康的肠道内环境对斑点叉尾鮰的肠道组织发育有促进作用。也有研究发现,仔猪饲料中添加120 μg/mL的海藻多糖可以显著上调小肠紧密连接蛋白Occludin、Claudin-1和ZO-1的mRNA相对表达量[24],紧密连接蛋白是肠道机械屏障的基础结构,海带酶解提取物可能通过促进紧密连接蛋白的表达来维持肠道机械屏障功能的完整性。

  • 肝脏代谢营养物质的能力对鱼体的生长性能和健康状态有极大地影响,因此分析不同表型的肝脏转录组有助于阐明海带酶解提取物促生长的分子作用机制。不同表型的组织基因表达量的差异也是巨大的,而其中差异最为显著的基因可能就是作用靶点之一。依据实验结束时斑点叉尾鮰肝脏转录组分析的结果,筛选出了其中上下调差异最明显的前10位基因,分析结果如下。

    NCOA2是p160类固醇受体共激活因子家族主要成员之一。在脂肪细胞中,NCOA2作为共激活因子与脂肪细胞分化转录因子-过氧化物酶体增生物PPARG相互作用,通过减弱Phospho-PPARG-S114复合物来促进脂肪细胞的分化,增加脂肪细胞的数量。因此NCOA2对脂肪沉积有重要的调控作用[25]。本实验中NCOA2基因高表达对鱼体脂肪沉积有一定的积极作用。

    KLF12是一种与真核细胞转录调控相关的锌指蛋白,研究证实其与脂代谢的调节也有相关作用,可以显著促进脂肪酸合成酶(FAS)、乙酰CoA羧化酶(ACC)等脂代谢相关基因的表达水平提高。此外,脂蛋白脂肪酶相关基因表达也显著提高。同时还推测KLF12基因在改善脂肪肝、增强葡萄糖耐受性以及增加胰岛素敏感性方面有重要作用[26]

    在上调的差异基因中,描述为维生素D3羟化酶相关蛋白样亚型X1和C-C基序趋化因子样21a两种基因,其GO功能注释均与细胞膜相关。维生素D3羟化酶的生物学作用之一是促进小肠黏膜对钙和磷的吸收[27]。C-C基序趋化因子则与免疫球蛋白A(IgA)的合成相关,肠道黏膜产生的IgA能阻止毒素以及抗原入侵,其免疫效应对肠道健康起到重要的保护作用[28]。从上述结果来看,海带酶解提取物对于肠黏膜的吸收作用以及维持肠道健康有积极影响。

    日粮添加海带酶解提取物后,肝脏转录组结果显示,差异表达基因显著富集在12条KEGG通路中,这些信号通路主要涉及在细胞增殖与分化、脂类代谢、糖类代谢以及生长因子代谢、免疫等方面。其中,G6PC基因涉及其中6条信号通路,占总数的二分之一,可见该基因很可能是影响多种代谢的潜在靶位点之一。G6PC即6-磷酸葡萄糖酶催化亚基,是6-磷酸葡萄糖酶(G6Pase)的组成部分,在机体内作用位催化6-磷酸葡萄糖水解生成葡萄糖和磷酸,是糖原分解和糖异生的最后一步反应[29-30]。研究表明,营养素可以调节鱼类肝脏中的G6PC水平,高糖饲料投喂草鱼(Ctenopharyngodon idella)后G6PC表达量上调[31]。本实验结果显示,日粮添加海带酶解提取物后饲料成分改变,肝脏G6PC水平显著升高。此外,激素也对鱼类的G6PC表达水平有调控作用,研究发现胰岛素可以抑制虹鳟(Oncorhynchus mykiss)的G6PC表达水平[32]。在本实验条件中,添加剂用量极低,含糖量变化幅度较小,并且KEGG通路有显著富集在胰岛素信号通路上的现象。而已有研究表明,海带酶解提取物的主要成分海带多糖可以提高胰岛素水平[33]。因此海带酶解提取物可能是通过激素如胰岛素调控G6PC的表达水平,最终调控代谢酶的活性促进鱼体生长。

    转录组实验结果主要用于揭示饲料添加海带酶解提取物后对斑点叉尾鮰生理代谢和生理机能的影响。本实验结果显示,饲料中海带酶解提取物的确对斑点叉尾鮰的生理代谢产生了积极影响,尤其是在作为信号分子的作用机制方面有增强的作用。饲料中的海带酶解提取物可能通过影响生理代谢信号分子与激素调控作用、影响脂代谢和能量代谢等途径促进斑点叉尾鮰的生理代谢和生长,并对肠道黏膜、肝脏等组织结构产生了积极影响。在生理功能作用方面,显示出有增强免疫作用的能力,具体的作用位点、作用途径或作用方式等还需要进一步的研究。

4.   结论
  • 本实验条件下,在初始体质量为(51.18±1.14) g的斑点叉尾鮰日粮中添加海带酶解提取物可以提高其生长速率,降低饲料系数,改善肠道形态结构。推荐斑点叉尾鮰饲料中海带酶解提取物添加量为0.96~0.98 g/kg。

    肝脏转录组的结果显示,适宜添加量的海带酶解提取物对斑点叉尾鮰肝脏的糖脂代谢水平有积极影响。

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