侏儒型华南鲤的生长和摄食性能分析及vegf121基因的表达特征

Growth performance, feed intake and vegf121 gene expression feature of dwarf Cyprinus carpio rubrofuscus

关键词:  华南鲤  血管内皮生长因子  侏儒  摄食

Key words:  Cyprinus carpio rubrofuscus  Vascular endothelial growth factor  Dwarf  Feed intake

摘要:

为了探究侏儒型华南鲤(Cyprinus carpio rubrofuscus)生长发育迟缓的分子机制，深入分析血管内皮生长因子(vascular endothelial growth factor, VEGF)在鱼类生长过程中的作用，本研究比较分析了侏儒型华南鲤和生长正常华南鲤(对照组)的生长性状、摄食率和饲料利用率等指标，并扩增和测序分析了华南鲤vegf121基因的cDNA序列，利用实时荧光定量PCR方法(qRT-PCR)检测了生长正常华南鲤vegf121基因的mRNA在11种组织中的表达量，以及在侏儒型鱼的心脏、垂体、大脑、肌肉和下丘脑中的表达水平。摄食实验结果显示，侏儒型华南鲤的摄食率和增重率显著低于体型相似的生长正常华南鲤，而饲料系数与正常鱼无显著差异。同时，获得华南鲤vegf121基因cDNA序列1 235 bp，其中，开放阅读框432 bp，编码144个氨基酸。该蛋白属于VEGF家族的VEGF121亚型。其氨基酸序列与11种已知鱼类VEGF121序列同源性在79.86%以上，而与人类(Homo sapiens)和小鼠(Mus musculus)的同源性仅为51.02%和52.74%。VEGF121亚型在人类和鱼类的同源性较低且时空分布存在较大差异，可能与其呼吸方式和呼吸系统的血管形成模式差异有关。qRT-PCR结果显示，在生长正常华南鲤11个组织中均能检测到vegf121 mRNA的表达，说明vegf121对于华南鲤不同组织中血管的发育有不同程度的调控作用。比较侏儒型华南鲤和生长正常鱼的下丘脑等5个组织中该基因的表达发现，在侏儒型华南鲤下丘脑组织中的表达水平显著高于生长正常鱼；而在大脑和垂体中，vegf121的表达水平相当；在肌肉和心脏组织中，vegf121的表达水平都较生长正常鱼低，但差异不显著。因此，推测侏儒型华南鲤的生长发育缓慢可能与下丘脑的血管生成异常和器质性病变影响鱼体的食欲调控信号有关，进而导致鱼体生长缓慢。本研究为进一步探讨侏儒型华南鲤生长发育缓慢的原因提供了科学依据。对养殖经济鱼类群体中侏儒型个体产生原因的遗传学研究及增加养殖鱼类的产量具有重要意义。

Abstract:

To explore the molecular mechanism of dwarf fish formation in the cultured population of Cyprinus carpio rubrofuscus and the functions of vascular endothelial growth factor (VEGF) in fish growth, the features of growth trait, feed intake, and feed utilization of dwarf fish were detected and analyzed. Vegf121 cDNA was amplified and sequenced in this study. The expression levels of vegf121 were analyzed by quantitative real time PCR (qRT-PCR) in 11 tissues of normal fish, and in the heart, pituitary, brain, muscle, and hypothalamus of dwarf fish. The results showed that under the culture condition in ponds, the body length distribution of the typical five-month-old C. carpio rubrofuscus population with dwarf individuals showed two obvious independent normal distribution curves. Furthermore, the dwarf individuals formed a small population that distinguished it from the normal and fast-growing fish in terms of body size. The results of the feeding experiment showed that the daily feed intake (DFI) and the weight gain rate (WGR) of fish in the dwarf group were significantly lower than those in the normal group, whereas the feed conversion rate (FCR) showed no significant difference between the two groups. Meanwhile, vegf121 was found to be differentially expressed in C. carpio rubrofuscus. The partial cDNA sequence of vegf121 gene was 1,235 bp with an open reading frame of 432 bp encoding 144 amino acids. According to the predicted amino acid sequence, the protein belongs to the VEGF121 isoform and has a relatively high sequence identity to the VEGF121 proteins of 11 known fish with the lowest identity of 79.86% aligned to Denticeps clupeoides. However, the identities to VEGF121 isoforms to those of Homo sapiens and Mus musculus were lower, at only 51.02% and 52.74%, respectively. The low homology and different spatial and temporal distribution of VEGF121 isoforms between animals and fishes may be related to breathing regulators and the differences in vascularization patterns in the respiratory system. The qRT-PCR results of the expression levels of vegf121 mRNA detected in the tissues of the normal growth fish showed that the relative expression levels were the highest in the muscle, followed by those in the heart, gill, brain, stomach, pituitary, eye, liver, kidney and spleen, and the lowest in the hypothalamus. This suggested that vegf121 has different regulatory functions in the development of blood vessels in different tissues of C. carpio rubrofuscus. Comparing the expression of vegf121 in the five tissues between the dwarf and normal-growth fish group revealed that the expression levels of vegf121 mRNA in the hypothalamus of the dwarf fish group were significantly higher than those of the normal fish group. The hypothalamus, as a high-level nerve center regulating endocrine and metabolic activities in humans and fish, may undergo some pathological changes under the action of certain environmental or genetic factors. The higher expression of vegf121 in the hypothalamus may have disrupted hormone secretion in C. carpio rubrofuscus and subsequently destroyed the metabolic balance in the body, resulting in a decrease in the appetite and feeding ability of fish. However, there was no significant change in the expression of vegf121 mRNA in two other neural tissues, the brain and pituitary, between the two groups. While the expression levels of vegf121 in the both muscle and heart were slightly lower than those in the normal-growth group. The abnormally higher expression of vegf121 in the hypothalamus indicated angiogenesis or lesions in the hypothalamus, which may have affected the feed intake regulation pathway signals in the fish body. This study provides a scientific basis to further explore the mechanism leading to dwarfism in C. carpio rubrofuscus. Genetic studies on the reasons leading to dwarf individuals in cultured economic fish populations can help increase the yield and productivity in aquaculture.