Comparative analysis and evolutionary significance of the HMG1 gene in crucian carp,blunt snout bream,and their polyploid progeny

The full-length mRNA of the high mobility group protein 1 coding gene (HMG1) was obtained by RACE-PCR from red crucian carp (Carassius auratus red var.),blunt snout bream (Megalobrama amblycephala),and their triploid and tetraploid progeny.The sequence contained an open reading frame of 579 nucleotides coding for 193 amino acids.The nucleotide identity of HMG1 was higher between the tetraploid hybrid and the maternal red crucian carp (99%) than between the tetraploid hybrid and the paternal blunt snout bream (97%).The nucleotide identity between the triploid hybrids and each parent (95%) was lower than that between the parents (98%).The protein identity between the tetraploid hybrid and each parent (100%) was higher than that between the triploid hybrid and each parent (97%).Our results suggest that interspecific hybridization generates a shock to the HMG1 gene in triploid hybrids,causing divergence of nucleotides.The HMG1 protein of the tetraploid hybrids was consistent with that of its parents,which reduced the barrier of cross incompatibility between alleles,providing the basis for the bisexual fertile tetraploid hybrids forming a new polyploid species in nature.The secondary and tertiary structures of the HMG1 protein contain eight helices,three switches,two DNA-binding domains in the N-terminus,and a long acidic tail in the C-terminus.Together,these data suggest that the HMG1 protein plays a role of protein-DNA interactions,facilitating various DNA-dependent activities in the nucleus.We also investigated the phylogeny of fish,amphibian,reptilian,bird,and mammalian HMG1 proteins.Our results suggest that HMG1 is an ancestral protein that has been highly conserved.These data provide clues as to how interspecific hybridization may form polyploid hybrids.     

Maternal inheritance in polyploid fish inferred from mitochondrial ATPase genes analysis

The sequences of the ATPase8/6 genes for the triploid, tetraploid and pentaploid hybrids as well as for their male parent blunt snout bream were determined. In order to examine mitochondrial maternal inheritance, the sequences were subjected to a comparative sequence analysis with the homologous sequences of red crucian carp, their female parent, and zebrafish as the outgroup. Base composition and variation as well as the divergences based on nucleotide sequences and deduced amino acid sequences were calculated. Phylogenetic trees were also constructed with maximum parsimony (MP), minimum evolution (ME), neighbor joining (NJ) and the unweighted pair group method with arithmetic mean (UPGMA) algorithms in MEGA 3.1. The results showed that most nucleotide substitutions occurred at the third codon position of the two genes and thus represented synonymous mutations. The nucleotide sequence divergences of the ATPase8/6 genes ranged from 0.0% to 21.6% among ingroup samples (three types of polyploids and their parents), and 27.0–28.2% between their ingroup and the outgroup samples. All the polyploids were considerably closer in sequence relationship to the female parent red crucian carp (0.0–3.3%) compared to their male parent blunt snout bream (21.0–21.6%). The phylogenetic trees also showed a similar result. In conclusion, the mitochondrial ATPase8/6 genes of artificial polyploid fish stringently indicated maternal inheritance. Our results also suggested that the ATPase8/6 genes are valuable genetic markers to track genealogies and variations in the progenies of the hybrids.     

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.     

Rapid growth and sterility of growth hormone gene transgenic triploid carp

Triploid carp (100%) with 150 (3n=150) chromosomes were obtained by crossing the females of improved tetraploid hybrids (♀, 4n=200) of red crucian carp (♀)×common carp (♂) with the males of diploid yellow river carp (♂, 2n=100). The crosses yielded transgenic triploid carp (positive triploid fish, 44.2% of the progeny) and non-transgenic triploid carp (negative triploid fish). Histological examination of the gonads of 24-month-old positive triploid fish suggested they were sterile and the fish were not able to produce mature gametes during the breeding season. Morphologically, both the positive and negative triploid fish were similar. They had a spindle-shaped, laterally compressed, steel grey body with two pairs of barbells. Most of the quantifiable traits of the triploid carp were intermediate between those of the two parents. The positive and negative triploid fish were raised in the same pond for 2 years. The mean body weight of the positive triploid fish was 2.3 times higher than the negative triploid fish. The weight of the largest positive triploid fish was 2.91 times higher than that of the largest negative triploid fish. Thus, we produced fast-growing transgenic triploid carp that have a reduced ecological risk because of their inability to mate and produce progeny.     

基于DNA含量的复合鲫倍性分析

通过对复合鲫血细胞DNA含量的测定,探讨了其倍性和产生机制等方面的问题.结果显示,复合鲫的DNA含量明显多于母本种彭泽鲫的DNA含量,且所测14尾中多数个体的DNA含量超过或接近四倍体的DNA含量,少数个体的DNA含量介于三倍体和四倍体之间.推测多数复合鲫个体为复合四倍体异育彭泽鲫,其染色体组包含母本彭泽鲫的全套染色体和父本单倍染色体组所产生的子代,少数个体可能为非整倍体.     

五种淡水鱼对固定气泡幕反应初探

观察了尼罗罗非鱼(Tilapia nilotica),鲢(Hypophthalmichthys moli-trix)、鳙(Aristichthys nobilis)、草鱼(Ctenopharyngodon idellus)和鲫鱼(Caras-sius auratus)等五种淡水鱼对固定气泡幕的反应,气泡幕对这五种鱼均有一定的阻拦作用,但阻挡效果不理想,阻拦率分别为:罗非鱼53％,鲢44.6％,鳙27.6％,草鱼23.3％,鲫鱼21.7％,该五种鱼对气泡幕均有明显的适应现象,随着实验时间的延长和实验重复次数的增加,阻拦率常呈下降趋势。     

Evolutionary analysis of allotetraploid hybrids of red crucian carp × common carp, based on ISSR, AFLP molecular markers and cloning of cyclins genes

The allotetraploid hybrids of red crucian carp x common carp are the first reported artificially cultured polyploid fish with bisexual fertility and stable inheritance in vertebrate. Using ISSR and AFLP markers and the cyclins genes, the genomes and cyclin gene sequence changes were analyzed between the allotetraploid hybrids and their parents. The results indicated that the allotetraploids inherited many genetic characteristics from their parents and the genetic characteristics were stable after 15 generations. However, the allotetraploids had a closer genetic relationship with their original female parents and represented a bias toward the maternal progenitor. DNA fingerprinting analysis showed that the allotetraploids had undergone sequences deletion from their original parents and that the deleted sequences were mostly from the male parent＇s genome. Some non-parental bands were found in the allotetraploid hybrids. Sequences analysis of the cyclin A1 and B1 genes showed nonsynonymous substitutions of single nucleotides in codons that were different from their original parents, leading to non-parental amino acid loci. We speculate that the non-additivity in the allotetraploids, compared with their progenitors, could be an adjustment to the genomic shock from heterozygosity and polyploidy, allowing maintenance of genetic stability.     

137 Cs-γ 辐照对实验红鲫 AFLP 多态性的影响

摘要:目的 观察137 Cs-γ 辐照对实验红鲫 AFLP 的变化,探讨不同剂量137 Cs-γ 辐照对实验红鲫 DNA 多态性的影响。 方法 根据前期实验结果,将实验红鲫 C1HD 系随机分为 5 组,10 尾 / 组,即空白对照组、1 / 16 LD50 、1 / 8 LD50 、1 / 4 LD50 、1 / 2 LD504 个辐照组,设置 1. 94 Gy、3. 88 Gy、7. 76 Gy、15. 53 Gy4 个辐照剂量组和一个空白对照组,用生物辐照仪对实验红鲫进行一次性辐照。 提取辐照前和辐照后的实验红鲫血液 DNA,经 AFLP-PCR 扩增,并进行多态性信息含量( PIC) 、基因杂合度( H) 、香农信息指数( I) 分析。 结果 实验红鲫在137 Cs-γ 辐照处理前的 PIC、H 和 I分别为 45. 20% 、0. 19 和 0. 28,经137 Cs-γ 辐 照 处 理 后 分 别 为 72. 27% 、0. 28 和 0. 41,且 遗 传 学 指 数 升 高。 结 论经137 Cs-γ 辐照处理后,实验红鲫的 AFLP 多态性信息含量增加,基因杂合度升高。 AFLP 标记实验红鲫 C1HD 系可用于监测核辐射污染。     

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.     

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.     

Rapid establishment of pure lines of silver carp (Hypophthalmichthys molirix) and bighead carp (Aristichthys nobilis)

The diversity of gynogenetic, artificial sex reversal and natural silver carp and bighead carp is examined using randomly amplified polymorphic DNA (RAPD) method.All of the 187 bands are obtained and 19 (10.16%) of them are polymorphic in gynogenetic silver carp.Meanwhile 32 (15.61%) out of 205 bands are polymorphic in control group.In gynogenetic bighead carp a total of 232 bands are identified and 11 (4.74%) out of them are polymorphic, while 25 (10.37%) out of 241 bands are polymorphic in control group.The genetic distance of four populations is calculated and it is 0.102 and 0.023 for gynogenetic silver carp and gynogenetic bighead carp respectively.The values of natural silver carp and bighead carp are 0.161 and 0.104.From the UPGMA trees constructed based on genetic distance, the sex reversal individuals that match with the gynogenetic female individuals are picked out.A new breeding process of establishing a pure line is developed.     

红鲫与金鱼对洞庭湖外睾吸虫感染的对比实验

目的建立洞庭湖外睾吸虫的宿主动物模型。方法将红鲫与金鱼分别投放於含有洞庭湖外睾吸虫尾蚴钉螺的去氯自来水中自然感染,分阶段解剖观察。结果红鲫与金鱼对洞庭湖外睾吸虫尾蚴的感染率均为100%,并且尾蚴发育成成熟囊蚴。实验过程中,金鱼死亡3尾,而红鲫无一尾死亡。结论红鲫与金鱼均可作为洞庭湖外睾吸虫的宿主动物模型。红鲫作为洞庭湖外睾吸虫宿主动物模型更理想。     

实验红鲫水族箱饲养技术

目的 为完善实验红鲫标准化内容建立水族箱饲养技术。方法 总结多年来的实践经验,并参考文献。结果 选择大小约为1 200 mm×480 mm×755 mm(长×宽×高)的长方形水族箱,设置增氧泵、过滤器、照明灯等配套设施,模拟池塘生境进行布景。注入曝除氯气3 d后的自来水,保持水温在18～24 ℃,溶氧量在≥6 mg/L,酸碱度在pH7.5～8.5、光照在100～200lx、硬度(CaCO3)在95～105 mg/L、氨(NH3)浓度在≤0.02 mg/L等。按100 mm体长的80尾/m3水体的饲养密度,投放质量合格的实验红鲫。每日投喂全价颗粒饲料1～2次,每3～7 d部分换水1次,14～21 d全部换水并清洗水族箱1次,坚持做好日常管理与清洗消毒工作。结论 本文描述的饲养技术适合于用水族箱饲养实验红鲫。     

微卫星标记预警实验红鲫对苯酚遗传毒性响应的研究

摘要: 目的探讨微卫星标记预警实验红鲫对苯酚遗传毒性的响应。方法选择5 个微卫星标记,观察4 个苯酚 染毒组经8 d 染毒实验,苯酚对实验红鲫C1HD 系微卫星多态性的影响。结果苯酚在实验红鲫C1HD 系体内的 富集提高了基因杂合度( H) 、基因多态性信息含量( PIC) 等群体遗传学指数。在1 /8 LC50苯酚浓度组染毒第6 天,基因杂合度( H = 0. 15 ± 0. 19) 和基因多态性信息含量( PIC = 0. 42) 均达到峰值。结论体内富集一定浓度的苯酚可致实验红鲫C1HD 系微卫星多态性改变。微卫星基因杂合度( H) 和多态性信息含量( PIC) 随苯酚染毒时间的延长和富集量的增加而升高。     

四川黑山羊品种(群体)的RAPD标记研究

从5组24个随机引物中筛选出16个重复性好的多态引物,对四川9个黑山羊品种(群体)共计572只个体,进行随机扩增多态DNA(RAPD)标记研究.结果表明,16个引物扩增出125条带,其中102条带呈现多态,多态率为81.61%.不同引物扩增出的DNA片段在各品种(群体)中的分布频率不同.9个黑山羊品种(群体)间相似系数为0.7533～0.9642,遗传距离指数为0.0358～0.2467.引物OPQ-06(序列为GAGCGCCTTG)未在乐至黑山羊中扩增出900bpDNA片段,而在其他8个黑山羊品种(群体)中出现率均为1,可作为区分乐至黑山羊和其他8个黑山羊品种(群体)的分子遗传标记.引物OPK-03(序列为CCAGCTTAGG)未在江安黑山羊扩增出550bpDNA片段,在其他8个黑山羊品种(群体)中出现率均为1,可用于区分江安黑山羊和其他8个黑山羊品种(群体)的分子遗传标记.     

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…     

牛结核分支杆菌临床分离株rpsL，rpoB，KatG基因突变分析

采用噬菌体生物扩增法对本实验室分离鉴定的25株奶牛结核分支杆菌进行药敏分析,检测出14株耐链霉素、利福平、异烟肼菌株．用PCR扩增耐药株的rpsL基因片段（370bp）、rpoB基因片段（213bp）和KatG基因片段（458bp）,并对目的片段进行测序分析,其中5株耐链霉素菌株的rpsL基因在43位点发生突变,均由赖氨酸密码子（AAG）突变为精氨酸密码子（AGG）;5株利福平耐药株的rpoB基因在531位点、526位点、5¨位点发生突变,其中有3株分离株的rpoB基因在531位点发生点突变,1株分离株的rpoB基因在526位点发生突变,1株分离株的rpoB基因在531位点和511位点发生了较为少见的联合突变;4株异烟肼耐药菌株,其中2株在315位点发生碱基突变,2株在463位点发生碱基突变．