Induction of gynogenesis in red crucian carp using spermatozoa of blunt snout bream

Gynogenetic diploid was induced in red crucian carp (RCC) ( Carassius auratus Red Variety) eggs using UV-irradiated spermatozoa from blunt snout bream (B) (Megalobrama amblycephaia) or from mirror carp (C) (Cyprinus carpio. L) . Spermatozoa were genetically inactivated by an appropriate UV dosage, and then the maternal DNA was duplicated with cold shock at 0-4℃. When using the spermatozoa of B, the fertilization rate, hatching rate and survival at first feeding were 52. 6±3. 0 %, 23.6±4.1 % and15.7±3.4 % , respectively, and the survival at first feeding was significantly higher than that (11.3±2.2%) when using the spermatozoa of C (Cyprinus carpio. L). According to the morphological characteristics, the chromosome number and the degree of gonadal development, gynogenetic RCC could be distinguished from the control hybrids of RCC♀×B♂. The individuals with red body color, 100 chromosomes and normal gonadal development were successful gynogenetic RCC, while the individuals with 124 or 148 chromosomes and delayed gonadal development were hybrids of (RCC×B). The triploid hybrids (RCC×B) (2 years old) were sterile, but the tetraploid hybrids (RCC×B) were sexually mature age of two. In the present study, compared to the spermatozoa of C, the advantages of spermatozoa of B as the activation source were that could increase the survival at first feeding of gynogenetic individuals and simplify the confirmation of gynogenetic status, which suggested that the sperm of B was an effective activation source for inducing gynogenesis in crucian carp.     

Analysis of intron sequence variability of the conservative HMG-box of Sox9 genes in allotetraploids and their original parents

The Sox genes of allotetraploids and their original maternal red crucian carp (Carassius caassius red var.) and original paternal common carp (Cyprinus carpio L.) were detected by PCR with the designed primers based on the conserved HMG-box sequence in different species. Sequencing of Sox genes indicated that two Sox9 genes (Atsox9a and Atsox9b) existed in allotetraploids, while only one Sox9 gene existed in red crucian carp (Rcsox9a) and common carp (Ccsox9b) . All of the four Sox9 genes contained an intron in the HMG-box. with the sizes of 413 bp, 703 bp, 401 bp and 714 bp, respectively. Moreover, the introns obeyed the rule of "GT-AG" . A high similarity was observed between introns of Atsox9a and Rcsox9a (94.4%), Atsox9b and Ccsox9b (97.8%). Interestingly, the deduced amino acid sequences of their corresponding exons all shared 100% identity. Thus, introns of the HMG-domain of Sox9s in allotetraploids and their original parents have not only the length polymorphism but also intron variability. Our results provide significant molecular evidence for the origin and evolution of allotetraploids.     

Induction of gynogenesis in red crucian carp using spermatozoa of blunt snout bream

Gynogenetic diploid was induced in red crucian carp (RCC) (Carassius auratus Red Variety) eggs using UV-irradiated spermatozoa from blunt snout bream (B) (Megalobrama amblycephala) or from mirror carp (C) (Cyprinus carpio. L). Spermatozoa were genetically inactivated by an appropriate UV dosage, and then the maternal DNA was duplicated with cold shock at 0-4℃. When using the spermatozoa of B, the fertilization rate, hatching rate and survival at first feeding were 52.6±3.0 %, 23.6±4.1 % and 15.7±3.4 %, respectively, and the survival at first feeding was significantly higher than that (11.3±2.2%) when using the spermatozoa of C (Cyprinus carpio. L). According to the morphological characteristics, the chromosome number and the degree of gonadal development, gynogenetic RCC could be distinguished from the control hybrids of RCC♀× B♂. The individuals with red body color, 100 chromosomes and normal gonadal development were successful gynogenetic RCC, while the individuals with 124 or 148 chromosomes and delayed gonadal development were hybrids of (RCC× B). The triploid hybrids (RCC× B) (2 years old) were sterile, but the tetraploid hybrids (RCC× B) were sexually mature age of two. In the present study, compared to the spermatozoa of C, the advantages of spermatozoa of B as the activation source were that could increase the survival at first feeding of gynogenetic individuals and simplify the confirmation of gynogenetic status, which suggested that the sperm of B was an effective activation source for inducing gynogenesis in crucian carp.     

Analysis of intron sequence variability of the conservative HMG-box of Sox9 genes in allotetraploids and their original parents

The Sox genes of allotetraploids and their original maternal red crucian carp （Carassius caassius red var.） and original paternal common carp (Cyprinus carpio L.) were detected by PCR with the designed primers based on the conserved HMG-box sequence in different species. Sequencing of Sox genes indicated that two Sox9 genes (Atsox9a and Atsox9b) existed in allotetraploids, while only one Sox9 gene existed in red crucian carp (Rcsox9a) and common carp (Ccsox9b). All of the four Sox9 genes contained an intron in the HMG-box, with the sizes of 413 bp, 703 bp, 401 bp and 714 bp, respectively. Moreover, the introns obeyed the rule of “GT-AG”. A high similarity was observed between introns of Atsox9a and Rcsox9a (94.4%), Atsox9b and Ccsox9b (97.8%). Interestingly, the deduced amino acid sequences of their corresponding exons all shared 100% identity. Thus, introns of the HMG-domain of Sox9s in allotetraploids and their original parents have not only the length polymorphism but also intron variability. Our results provide significant molecular evidence for the origin and evolution of allotetraploids.     

A new type of triploid crucian crap?red crucian carp (♀)×allotetraploid (♂)

A new type of triploid hybrids (TC) was produced by crossing red crucian carp (♀) with allotetraploid hybrids (♂). This type of triploids with no barbel was spindle-shaped and gray in color. Compared with their parents, some data of the morphological traits in the triploid hybrids were intermediate to those of their parents, and some were beyond their parents with heterozygous traits. Under the same culture conditions, the growth rate of the new type triploids was faster than their maternal red crucian carp (RC). The gonadal development of triploids was obviously slower than that of diploids, and the germ cells in them were degenerated. Compared with another type of triploids produced by Japanese crucian carp (♀) with allotetraploid hybrids (♂), the new type triploids not only kept faster growth rate and sterility, but also had no barbel that was similar to the common carp. The absence of the barbel in the new type of triploids has the important significance in both the inheritance and fish breeding.     

Molecular cloning and polymorphism of major histocompatibility complex class Ⅰ genes from grass carp (Ctenophayngodon idellus)

In order to clarify the molecular sequences, allelic polymorphism and the tertiary structure of grass carp(Ctenophayngodon idellus) MHC class Ⅰ, and to further study their relationship with disease resistances, grass carp MHC class Ⅰ gene (Ctid-MHC I) was cloned from a cDNA library and the allelic polymorphism in the population was investigated. The results showed that most of the variations exist in the peptide-binding domain (PBD) and high polymorphism was identified in the Ctid-MHC I allelic genes from 12 individuals. Based on the genetic distance, Ctid-MHC class Ⅰ can be classified into 6 types (from Ctid-MHC I-UA to Ctid-MHC I-UF) which were subdivided into 9 lineages (from A to I). Comparison of the Ctid-MHC I among animals and humans showed that the key amino acids of the peptide binding sites are conserved. Analysis of the tertiary structure of the PBD between Grass carp and human crystallographic data of HLA-A2, the variation with insertion or deletion was found in eight regions (A-H). The p     

Rapid growth cost in “all-fish” growth hormone gene transgenic carp:Reduced critical swimming speed

Evidence has accumulated that there is a trade-off between benefits and costs associated with rapid growth. A trade-off between growth rates and critical swimming speed (Ucrit) had been also reported to be common in teleost fish. We hypothesize that growth acceleration in the F3 generation of "all-fish" growth hormone gene (GH) transgenic common carp (Cyprinus carpio L.) would reduce the swimming abilities. Growth and swimming performance between transgenic fish and non-transgenic controls were compared. The results showed that transgenic fish had a mean body weight 1.4―1.9-fold heavier,and a mean specific growth rate (SGR) value 6%―10% higher than the controls. Transgenic fish,however,had a mean absolute Ucrit (cm/s) value 22% or mean relative Ucrit (BL/s) value 24% lower than the controls. It suggested that fast-growing "all-fish" GH-transgenic carp were inferior swimmers. It is also supported that there was a trade-off between growth rates and swimming performance,i.e. faster-growing individuals had lower critical swimming speed.     

Prediction of a common neutralizing epitope of H5N1 avian influenza virus by in silico molecular docking

The H5N1 avian influenza virus (AIV) has widely spread in Asia, Europe and Africa, making a large amount of economic loss. Recently, our research group has screened a common neutralizing mono- clonal antibody named 8H5, which can neutralize almost all H5 subtype AIV ever isolated so far. Obvi- ously, this monoclonal antibody would benefit for research and development of the universal AIV vac- cine and design of the drug against H5N1 AIV in high mutation rate. In this study, the homology mod- eling was applied to generate the 3D structure of 8H5 Fab fragment, and "canonical structure" method was used to define the specified loop conformation of CDR regions. The model was subjected to en- ergy minimization in cvff force field with Discovery module in Insight II program. The resulting model has correct stereochemistry as gauged from the Ramachandran plot calculation and good 3D-structure compatibility as assessed by interaction energy analysis, solvent accessible surface (SAS) analysis, and Profiles-3D approach. Furthermore, the 8H5 Fab model was subjected to docking with three H5 subtype hemagglutinin (HA) structures deposited in PDB (ID No: 1jsm, 2ibx and 2fk0) respectively. The result indicates that the three docked complexes share a common binding interface, but differ in bind- ing angle related with HA structure similarity between viral subtypes. In the light of the three HA inter- faces with structural homology analysis, the common neutralizing epitope on HA recognized by 8H5 consists of 9 incontinuous amino acid residues: Asp68, Asn72, Glu112, Lys113, Ile114, Pro118, Ser120, Tyr137, Tyr252 (numbered as for 1jsm sequence). The primary purpose of the present work is to provide some insight into structure and binding details of a common neutralizing epitope of H5N1 AIV, thereby aiding in the structure-based design of universal AIV vaccines and anti-virus therapeutic drugs.     

鲤NADH泛醌氧化还原酶亚基3基因的克隆及低温适应相关性分析

根据鲤低温下特异表达的基因（片段）序列设计引物,采用2轮菌落PCR方法对鲤脑全长cDNA文库中的克隆进行筛选,确定了基因序列152在文库中的位置,经生物信息学比对分析确定其为NADH泛醌氧化还原酶亚基3基因.这一基因在低温下的特异表达,对于低温下鱼类机体的供能,起着重要作用.结果还表明,NADH泛醌氧化还原酶亚基3基因在鲤氧化呼吸链电子传递系统中与低温适应性状在一定程度上存在相关性.     

鲤IGF2b基因内含子的克隆、基因组序列分析及慢病毒载体构建

 为了了解鲤IGF2b基因与鲤生长性状之间的关系,以建鲤为试验材料,克隆了IGF2b基因内含子,分析其基因组序列的特点,构建了IGF2基因的慢病毒载体,同时观察其在293T细胞中的表达活性。结果获得鲤IGF2b 5 173 bp长度的基因组DNA序列(HM755899),共有3个内含子,4个外显子;在3′非翻译区存在2个CpG岛,在5′端非翻译区存在(T)n重复序列;除此之外,成功构建了慢病毒载体质粒Lenti-IGF2b-IRES-EGFP,转染293T细胞后,产生的重组慢病毒颗粒出现高表达绿色荧光,荧光定量PCR检测发现IGF2b基因在293T细胞中高表达。这些结果将为研究IGF2b基因在多态性、表达方面与鲤生长性状之间的关联奠定基础。     

Molecular cloning of growth hormone receptor (GHR) from common carp(Cyprinus carpio L.)and identification of its two forms of mRNA transcripts

Growth hormone receptor (GHR) belongs tothehematopoietic receptor superfamily[1]. The action ofgrowth hormone (GH) in regulating growth[2],re-production[3]and i mmunity[4]has been elucidated.The binding of GHtothe GHRontarget tissues trig-gers a cascade of tyrosine and protein phosphorylationevents, which cul minates in the biological action ofGH[5 ,6]. Up to date GHR cDNAs have been clonedfrom many species[7—9],including various kinds ofmammalian ani mals ; avian of chicken and domest…     

建鲤品种特性研究进展

建鲤是遗传性状比较稳定的人工育成的鱼类品种,生长快,体型体色美观,肉质好,适应性和抗病力强,生产性能优良.深入研究建鲤的形态特征、经济性状和遗传性能,保持建鲤优良的遗传特性,发挥其品种优势,扩大生产应用规模.     

麦饭石、稀土作为鲤鱼、皱纹盘鲍饲料添加剂研究

以麦饭石、麦饭石稀土作为鲤鱼、皱纹盘饱饲料中微量元素添加剂,与无机盐添加剂进行了对比喂养试验。结果表明：饲料中添加1％麦饭石能显著地促进鲤鱼和皱纹盘鲍幼鲍的生长;方差分析表明,在鲤鱼饲料中添加麦饭石的三个试验组鱼体平均增重率与无机盐对照组差异显著（P＜0．05）,与未添加微量元素添加剂的基础组差异极显著（P＜0．01）。皱纹盘鲍饲料中添加1％麦饭石的试验组鲍体平均增重率与无机盐对照组差异不显著,但与未添加微量元素的基础组差异显著（P＜0．05）。     

Acute and chronic un-ionized ammonia toxicity to ‘all-fish’ growth hormone transgenic common carp (<Emphasis Type="Italic">Cyprinus carpio</Emphasis> L.)

Ammonia is toxic to fish in natural and artificial waters. We evaluated the acute (96 h) and chronic (21 d) toxicity of un-ionized ammonia to GH transgenic common carp (Cyprinus carpio L.) and non-transgenic common carp using a static-renewal bioassay. The 24, 48, 72 and 96 h median lethal concentrations (LC₅₀) of un-ionized ammonia were slightly lower in transgenic carp (2.64, 2.44, 2.28 and 2.16 mg N/L, respectively) than in non-transgenic carp (2.70, 2.64, 2.52 and 2.33 mg N/L, respectively). Similarly, the median lethal time (LT₅₀) was significantly shorter for transgenic carp (1.41, 7.91 and 117.42 h) than for non-transgenic common carp (2.53, 14.06 and 150.44 h) following exposure to 3.86, 3.29, or 2.09 mg N/L, respectively. Moreover, the mortality of transgenic carp was significantly higher than that of non-transgenic carp at all un-ionized ammonia concentrations ((0.91 ± 0.12), (0.48 ± 0.06) and (0.12 ± 0.01) mg N/L) during the 21 d chronic toxicity test. Our results suggest that GH transgenic carp are less tolerant of un-ionized ammonia than non-transgenic carp. Our data are useful for evaluating potential environmental risk, optimizing stocking density in intensive aquaculture and establishing water quality criteria for ammonia in aquaculture.     

黄河鲤和德国镜鲤及其杂种F1同工酶的比较研究

本研究用垂直板聚丙烯酰胺凝胶电泳技术，以亲本黄河鲤（ＣｙｐｒｉｎｕｓｃａｒｐｉｏＬ．（ＹｅｌｌｏｗＲｉｖｅｒｃａｒｐ））和德国镜鲤（ＳｅａｔｔｅｒｅｄＣｙｐｒｉｎｕｓＣａｒｐｉｏ．Ｌ．ｍｉｒｒｏｒ）及其杂种Ｆ＿１的肝脏、脾脏、心、肾脏、脑和肌肉为材料，进行了乳酸脱氢酶同工酶（ＬＤＨ），苹果酸脱氢酶同工酶（ＭＤＨ）和酯酶同工酶（ＥＳＴ）电泳图谱的比较分析，结果表明，同种鱼不同组织的同工酶谱有明显的组织特异性，亲本及Ｆ＿１的同种不同组织同工酶谱也有差异，亲本各组织的大多数同工酶在Ｆ＿１代得到表观，杂种Ｆ＿１的同工酶谱的表现又可分为三类：１）与双亲完全互补型；２）部分互补和少数亲本酶带消失型；３）产生双亲均无得以出现的酶带或杂种酶带型．本文还讨论了黄河鲤、德国镜鲤及其Ｆ＿１的同工酶的遗传基础和亚基组成．     

稀土元素镧对鲤生长的影响

用含镧饵料（Ｌａ２Ｏ３）饲喂鲤（Ｃｙｐｒｉｎｕｓｃａｒｐｉｏ）能明显增进鲤的食欲、提高生长速度，对鲤的肝肾指数亦有明显的提高作用。实验发现镧元素在饵料中的含量为２０ｍｇ／ｋｇ时，生长效果最为明显。