Study on muscle fiber characteristics, metabolic enzymes and gene expression of caudal stalk of golden-backed carp (Cyprinus carpio var. Jinbei)
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摘要: 稻田金背鲤 (Cyprinus carpio var. Jinbei) 是多个世代经过长期自然选择和人工选择生活于稻田的鲤鱼。为推广金背鲤—稻田养殖模式，为该模式下的良种选育提供数据支撑，以池塘养殖的金背鲤为对照组，采用组织学、酶学及分子生物学方法分析了其尾柄肌纤维及相关代谢酶与基因表达，探究了稻田金背鲤尾柄肌肉在稻田浅水生态条件下生长的适应性特征。结果显示：在水稻扬花期和渔获期，尾柄肌纤维直径稻田组均大于池塘组并存在显著性差异 (P<0.05)；在水稻扬花期和渔获期，尾柄肌纤维横截面积稻田组大于池塘组，在渔获期存在显著性差异 (P<0.05)；尾柄肌纤维密度稻田组均小于池塘组，在渔获期存在显著性差异 (P<0.05)；在水稻扬花期和渔获期，代谢酶活中的乳酸脱氢酶 (LDH)、过氧化氢酶 (CAT) 和Ca2+-ATP酶活性稻田组均高于池塘组；在水稻扬花期，稻田组的过氧化物酶体增殖激活受体γ辅助激活因子α (PGC-1α) 基因表达量显著高于池塘组 (P<0.05)，而稻田组的腺苷酸活化蛋白激酶 (AMPK)、沉默信息调节因子1 (SIRT1) 基因表达量在水稻扬花期和渔获期均显著低于池塘组 (P<0.05)。研究揭示，稻田金背鲤尾柄肌肉有更大的肌纤维直径和横截面积，尾柄肌肉呈现较高的LDH和Ca2+-ATP酶活性，这是其适应稻田浅水生活的特点；稻田金背鲤尾柄抗阻运动会引起肌肉肥大，与其激活蛋白质合成通路有关，而池塘金背鲤通常进行长时间的耐力运动，与其线粒体生物合成有关；在水稻扬花期，稻田金背鲤PGC-1α表达量较高，可能与此阶段水稻开花、稻田中昆虫增多、生物多样性增加以及生物量增加有关。Abstract: Golden-backed carp (Cyprinus carpio var. Jinbei) is a common carp that has lived in rice fields for many generations through long-term natural and artificial selection. In order to promote its paddy field breeding model and provide data supports for the breeding of improved varieties by this model, we analyzed the caudal stalk muscle fibers, related metabolic enzymes and gene expressions by using histology, enzymology and molecular biology methods, and explored the adaptive characteristics of caudal stalk muscle growth under the shallow ecological conditions of rice field. The results show that the diameter of caudal stalk muscle fiber in paddy field group was larger than that in pond group at flowering stage and harvest stage with significant differences (P<0.05). The cross-section area of caudal stalk muscle fiber in paddy field group was larger than that in pond group at flowering stage and harvest stage, with significant differences at harvest stage (P<0.05), while the density of caudal stalk muscle fiber in paddy field group was smaller than that in pond group. There were significant differences at harvest stage (P<0.05). The activities of lactate dehydrogenase LDH, catalase CAT and Ca2+-ATPase in metabolic enzymes were higher in paddy field group than in pond group at flowering stage and harvest stage. The expression level of peroxisome proliferation-activated receptor γ-coactivator α (PGC-1α) gene in paddy field group was significantly higher than that in pond group (P<0.05). The results reveal that the expression levels of adenylate activated protein kinase (AMPK) and silence-regulating factor 1 (SIRT1) gene in paddy field group were significantly lower than those in pond group at flowering stage and harvest stage (P<0.05). The resistance movement of the caudal stalk of golden-backed carp in rice field caused muscle hypertrophy, which was related to the activation of protein synthesis pathway, while the endurance exercise, which was related to the mitochondrial biosynthesis. The higher PGC-1α expression level of golden-backed carp in rice flowering stage may be related to the increase of rice flowering, insects, biodiversity and biomass.
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图 1 耐力运动激活能量感应网络AMPK/SIRT1 /PGC-1α信号通路
Figure 1. Endurance exercise activates AMPK/SIRT1 /PGC-1α signaling pathway in energy sensing network
图 2 稻田和池塘金背鲤尾柄肌纤维HE染色图
注：a. 稻花前稻田金背鲤尾柄肌纤维 (200×)；b. 水稻扬花期稻田金背鲤尾柄肌纤维 (200×)；c. 水稻扬花期稻田金背鲤尾柄肌纤维 (400×)； d. 渔获期稻田金背鲤尾柄肌纤维 (200×)；e. 水稻扬花同时期池塘金背鲤尾柄肌纤维 (200×)；f. 渔获期池塘金背鲤尾柄肌纤维 (200×)。
Figure 2. HE staining of muscle fibers of caudal stalk of golden-backed carp in paddy fields and ponds
Note: a. Caudal stalk muscle fibers of golden-backed carp before rice flower (200×); b. Caudal stalk muscle fibers (200×) of golden-backed carp in rice field during flowering period; c. Caudal stalk muscle fibers (400×) of golden-backed carp in rice field during flowering period; d. Caudal stalk muscle fibers (200×) of golden-backed carp in paddy field; e. Caudal stalk muscle fibers of pond golden-backed carp (200×) at the same period of rice flowering; f. Caudal stalk muscle fibers of pond golden-backed carp (200×) during the harvest stage.
图 3 稻田和池塘金背鲤生长过程中主要代谢酶活性变化
注：1. 稻花前期；2. 水稻扬花期；3. 渔获期；每个数据表示3个重复；不同的小写字母表示相同组别 (稻田组)不同采样时间点的酶活性差异显著 (P<0.05)，*表示不同处理组在相同采样点上有显著性差异 (P<0.05)。
Figure 3. Changes of main metabolic enzymes during growth of golden-backed carp in paddy fields and ponds
Notes: 1. Before rice flowering period; 2. Flowering period of rice; 3. Harvest period; values are means (N=3). Different letters indicate a significant difference (P<0.05) among each growth period in rice field. *. Significant difference (P<0.05) between the two groups at the same sampling site.
图 4 稻田金背鲤生长过程中主要代谢酶活性变化
注：1. 稻花前期；2. 水稻扬花期；3. 渔获期；不同小写字母表示稻田组在不同生长时间点基因表达量差异显著 (P<0.05)。
Figure 4. Changes of main metabolic enzyme activities of golden-backed carp during growth
Note: 1. Before rice flowering period; 2. Flowering period of rice; 3. Harvest period. Different letters indicate significant differences (P<0.05) in rice field in each growth period.
图 5 稻田和池塘养殖的金背鲤不同生长时期AMPK、SIRT1和PGC-1α基因的相对表达量
注：1. 稻花前期；2. 水稻扬花期；3. 渔获期；每个数据表示 3 个重复；不同的小写字母表示稻田组但不同生长时间点基因表达量差异显著 (P<0.05)，而*表示不同处理组在相同采样点上有显著差异 (P<0.05)。
Figure 5. Relative expression of AMPK, SIRT1 and PGC-1α gene in different growth period of golden-backed carp in paddy field and pond
Note: 1. Before rice flowering period; 2. Flowering period of rice; 3. Harvest period. Values are X±SE (N=3). Different letters indicate significant differences in rice field in each growth period (P<0.05) . *. Significant difference between the two groups at the same sampling time (P<0.05).
表 1 本实验所需引物
Table 1. Primers for this study
引物
Primer序列 (5'—3')
Sequence (5'–3')长度
Length/bp温度
Temperature/ ℃应用
ApplicationAMPK-F CGGAGACACACTAGGAGTGG 141 60 qPCR AMPK-R ATCTCGCGTCGAATCTTCCC 141 60 qPCR PGC-1α-F TCTCCATACTCCCGCTCCG 230 60 qPCR PGC-1α-R TCAGCCCTCTCACATTCTCGTT 230 60 qPCR SIRT1-F GGCAGCTATGGTCAGCCTAC 135 60 qPCR SIRT1-R TGCTTGAGGACAGGACTTACAC 135 60 qPCR β-actin-F AAAACCAACCATGTGCGACG 238 60 qPCR β-actin-R CCGTGCTCAATGGGGTACTT 238 60 qPCR 
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表 2 金背鲤尾柄肌纤维直径
Table 2. Muscle fiber diameter of caudal stalk of golden-backed carp μm
采样时期
Sampling
period稻田实验组
Paddy field
experimental group池塘对照组
Pond control
group稻花前
Before rice flower31.88±4.41a 31.88±4.41A 水稻扬花期
Flowering period54.88±10.31b* 41.68±4.42B 渔获期
Harvest stage62.98±4.92bc* 46.00±2.69BC 注：同一列不同上标字母表示数据间有显著性差异 (P<0.05)；*表示同一行数据间有显示性差异 (P<0.05)；后表同此。 Note: Different superscript letters within the same column indicate significant differences between the data (P < 0.05); *. Significant difference between the data within the same row (P < 0.05). The same case in the following tables.
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表 3 金背鲤尾柄肌纤维横截面积
Table 3. Cross-sectional area of muscle fibers of caudal stalk of golden-backed carp μm2
采样时期
Sampling
period稻田实验组
Paddy field
experimental group池塘对照组
Pond control
group稻花前
Before rice flower1 065.99±549.36a 1 065.99±549.36 水稻扬花期
Flowering period1 711.76±748.25a 1 327.79±600.17 渔获期
Harvest stage2 845.43±70.82b* 1 360.62±393.08
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表 4 金背鲤尾柄肌纤维密度
Table 4. Muscle fiber density of caudal stalk of golden-backed carp 根·mm−2
采样时期
Sampling
period稻田实验组
Paddy field
experimental group池塘对照组
Pond control
group稻花前
Before rice flower1252.56±916.11 1252.56±916.11 水稻扬花期
Flowering period702.45±406.78 845.10±306.00 渔获期
Harvest stage351.59±8.88* 781.40±244.04
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