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Induction and identification of gynogenesis in Nibea albiflora 白花Nibea albiflora雌蕊发生的诱导与鉴定
Q4 Environmental Science Pub Date : 2013-01-01 DOI: 10.3724/SP.J.1231.2013.38437
Yukai Yang, Yangjie Xie, Mingyi Cai, Linjiang Jian, Qingkai Chen, Bao Lou, Zhiyong Wang
In order to establish a procedure to induce artificial gynogenesis for genetic improvement and sex control studies in Nibea albiflora,ultraviolet irradiation was used to genetically inactivate the sperm and cold shock was used to induce the chromosome duplication of the eggs.Relative DNA content in newly-hatched larvae of candidate haploid gynogens,candidate diploid gynogens and the control groups from normal fetilization were mensurated,and paternity test was made using SSR markers for confirmation of gynogenesis.The gynogenetic diploid was successfully induced by activating egg development with UV-irradiated sperm combined with cold shock to prevent extrusion of the second polar body.UV irradiation time range was preliminarily determined by an insemination trial with the sperms irradiated by different doses of UV.The fertilized egg hatchability exhibited typical Hertwig effect when sperm irradiation time was 0-100 seconds at a UV intensity of 3 800 μW/(cm2·s).All newly-hatched larvae exhibited typical haploid syndrome when the UV irradiation time was over 60 seconds.In addition,we designed an orthogonal experiment to further optimize UV irradiation time,cold shock initiation time and cold shock duration time.The orthogonal experiment results showed that the hatching rate was highest(16%)in the following conditions:sperm irradiation time was 60 seconds,cold shock temperature was 3-4 ℃,cold shock initiation time was 2 minutes after fertilization and cold shock duration time was 10 minutes.The larvae of the optimal group have the same morphological characteristics and cellular DNA content with normal diploids.A further paternity test with five compatible SSR primers of Larimichthys crocea proved that the larvae in the optimal group were all gynogenetic diploids without paternal specific alleles.
为了建立一种人工诱导雌雄同体的方法,对白鹭进行遗传改良和性别控制研究,采用紫外线照射法使精子基因失活,冷休克法诱导卵子染色体复制。测定了正常受精的候选单倍体、候选二倍体和对照组新孵化的幼虫的相对DNA含量,并利用SSR标记进行亲本鉴定,确认了雌性的发生。用紫外线照射的精子,结合冷激,激活卵子发育,防止第二极体的挤压,成功地诱导了雌性二倍体。通过不同剂量紫外线照射精子的授精试验,初步确定了紫外线照射时间范围。在3 800 μW/(cm2·s)紫外照射下,精子辐照时间为0 ~ 100 s,受精卵孵化率呈现典型的Hertwig效应。当紫外线照射时间超过60秒时,所有新孵化的幼虫都表现出典型的单倍体综合征。此外,我们还设计了正交实验,进一步优化紫外线照射时间、冷冲击起始时间和冷冲击持续时间。正交试验结果表明,在精子辐照时间为60秒,冷激温度为3 ~ 4℃,受精后冷激起始时间为2分钟,冷激持续时间为10分钟的条件下,孵化率最高(16%)。最佳组幼虫的形态特征和细胞DNA含量与正常二倍体相同。进一步用5个匹配的SSR引物进行亲本鉴定,结果表明最优组的幼鱼均为雌性二倍体,无父系特异性等位基因。
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引用次数: 4
The cross breeding and genetic analysis of hybrids of Larimichthys crocea(♀)and Nibea miichthioides(♂) 大鲵(♀)与大鲵(♂)杂交选育及遗传分析
Q4 Environmental Science Pub Date : 2013-01-01 DOI: 10.3724/SP.J.1231.2013.38438
Jian Linjiang, Yang Yukai, Liu Xiande, Chen Qing-kai, W. Zhiyong
To understand the feasibility of cross breeding between Larimichthys crocea ♀ and Nibea miichthioides ♂,two hybrid families were built(LN1 and LN2).Ploidy of F1 hybrids was identified.Moreover,ten microsatellite loci were used for genetic relationship analysis between F1 hybrids and their parents.The result showed that L.crocea(♀)and N.miichthioides(♂)can be successfully crossed and hybrid offspring of normal survival were produced.Survival rate of hybrid offspring at the age of 45 days post hatch reached about 30%.However,fertilization rate(29.0%,32.6%)and hatching rate(75.0%,76.7%)of two hybrid families were significantly lower than those in large yellow croaker families(P0.05).Appearance of juvenile hybrid fish is visibly different from those of large yellow croaker,which has characteristics of both parents:slender body,sharp blunt head and their body sides are covered with black-brown spots.The results of DNA relative content investigation and genetic relationship analysis demonstrated that more than 90% of the hybrid offspring were hybrid diploid,in addition,a small number of those were hybrid triploid and gynogenesis diploid.Till the age of 45 days,growth rate of both hybrid diploid and hybrid triploid was slower than that of large yellow croaker.These findings will provide reference for exploitation and management of F1 hybrids of L.crocea(♀)and N.miichthioides(♂).
为了解Larimichthys crocea♀与Nibea miichthioides♂杂交育种的可行性,构建了两个杂交家族(LN1和LN2)。鉴定了F1杂种的倍性。此外,利用10个微卫星位点对F1杂交种与亲本的亲缘关系进行了分析。结果表明,大菱鲆(♀)与大菱鲆(♂)可以成功杂交,产生了正常存活的杂交后代。杂交后代在孵化后45天存活率可达30%左右。两个杂交科的受精率(29.0%、32.6%)和孵化率(75.0%、76.7%)显著低于大黄鱼科(P0.05)。杂交幼鱼的外貌与大黄鱼明显不同,大黄鱼具有双亲的特征:身体细长,头尖钝,身体两侧布满黑褐色斑点。DNA相对含量调查和亲缘关系分析结果表明,杂交后代90%以上为杂交二倍体,少数为杂交三倍体和雌性二倍体。到45日龄,杂交二倍体和杂交三倍体的生长速度都比大黄鱼慢。本研究结果可为大角田鼠(♀)和小角田鼠(♂)F1杂交品种的开发和管理提供参考。
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引用次数: 3
Path analysis of effects of morphometric attributes on body weight of Exopalaemon carinicauda 形态计量学属性对河蟹体重影响的通径分析
Q4 Environmental Science Pub Date : 2013-01-01 DOI: 10.3724/SP.J.1231.2013.38465
Chengsong Zhang, Fuhua Li, J. Xiang
In order to ascertain the optimal and measurable indices used in artificial breeding of Exopalaemon carinicauda,the effects of morphometric attributes on body weight of E.carinicauda were analyzed by path analysis.Besides body weight,ten morphometric attributes,including the body length,total length,carapace length,carapace width,carapace height,first abdominal segment height,first abdominal segment width,abdomen length,number of upper frontal eminence spin and number of lower frontal eminence spin were recorded for 177 four-month-old E.carinicauda.The correlation coefficients among all attributes were calculated,and the path coefficients and determination coefficients were calculated by taking the body weight as a dependent variable,and the other ten morphometric attributes as independent variables in path analysis.The data indicated that the path coefficients of four morphometric attributes(body length,carapace width,carapace height,first abdominal segment width)to body weight reached significant difference(P0.01)level.Among above four significant factors for body weight,body length showed the greatest effect on body weight with the direct effect efficiency of 0.468* *.The direct effects of total length,carapace length,first abdominal segment height,abdomen length,number of upper frontal eminence spin and number of lower frontal eminence spin on body weight were quite low,therefore,they were regarded as insignificant factors influencing the body weight.The stepwise regression analysis established a multiple regression equation on the effects of body length,carapace width,carapace height,first abdominal segment width on body weight with partial regression coefficients of-3.277,0.470,0.980,0.528 and 1.942 separately.Judging from the high multiple-correlation coefficient(R2=0.976),the main attributes(body length,carapace width,carapace height,first abdominal segment width)determining the body weight have been selected.
采用通径分析的方法,分析了形态计量学属性对马爪鱼体重的影响,为确定马爪鱼人工养殖的最佳指标和可测量指标。除体重外,还记录177只4月龄马背天鼠体长、总体长、甲壳长、甲壳宽、甲壳高、第一腹段高、第一腹段宽、腹部长、上额隆起旋转次数和下额隆起旋转次数等10个形态计量学属性,计算各属性间的相关系数,并取体计算通径系数和决定系数在通径分析中,权重作为因变量,其他十个形态计量属性作为自变量。数据表明,4个形态计量属性(体长、甲壳宽度、甲壳高度、第一腹段宽度)与体重的通径系数均达到极显著水平(P0.01)。在上述4个影响体重的显著因素中,体长对体重的影响最大,直接影响效率为0.468* *。全身长度、甲壳长度、第一腹段高度、腹部长度、额上隆起旋转次数和额下隆起旋转次数对体重的直接影响均较低,认为对体重的影响不显著。逐步回归分析建立了体长、甲壳宽度、甲壳高度、第一腹段宽度对体重影响的多元回归方程,部分回归系数分别为-3.277、0.470、0.980、0.528和1.942。从较高的多重相关系数(R2=0.976)判断,选择了决定体重的主要属性(体长、甲壳宽度、甲壳高度、第一腹段宽度)。
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引用次数: 3
Molecular cloning and expression of allograft inflammatory factor 1 in Haliotis diversicolor under stresses 异色海胆同种异体移植物炎症因子1的克隆与表达
Q4 Environmental Science Pub Date : 2013-01-01 DOI: 10.3724/SP.J.1231.2013.38463
Yitao Huang, Xiuhong Cai, Ziping Zhang, Guodong Wang, Z. Zou, Shuhong Wang, Yilei Wang
Allograft inflammatory factor-1(AIF-1)is a kind of calcium-binding protein with EF-hand domain.AIF-1 mRNA is robustly induced by IFN-γ in murine macrophages.It is involved in transplant rejection,immune inflammatory reaction,non-inflammatory injury and so on.In this study,a molluscan AIF-1 gene,HdAIF-1,was cloned for the first time from Haliotis diversicolor.Its full-length cDNA sequence is 942 bp,with a 456 bp open reading frame encoding a protein of 151 aa.Quantitative real-time PCR results indicated that HdAIF-1 could be detected in all examined tissues,with the highest level in hemolymph and gill.Under thermal stress,HdAIF-1 was up-regulated in gill significantly at temperature-rise period and achieved the highest level when up to 31 ℃.However,the expression level of HdAIF-1 in hemolymph and hepatopancreas did not show significant difference between thermal group and control group from the first phase to the fourth,and its expression level was up-regulated significantly at 96 h in these two tissues.Under hypoxia stress,the expression level of HdAIF-1 in hemolymph showed no significant difference between control and exposed groups.However,it was down-regulated at 24 h and up-regulated at 192 h remarkably in gill.After Vibrio parahaemolyticus challenge,the HdAIF-1 expression level achieved the highest level at 3 h in hemolymph,reduced to the control level at 12 h,and then up-regulated significantly at both 24 h and 48 h.These results suggest that HdAIF-1 may play important roles as an immune factor under different stresses.
同种异体炎症因子-1(AIF-1)是一类具有EF-hand结构域的钙结合蛋白。在小鼠巨噬细胞中,IFN-γ强烈诱导AIF-1 mRNA表达。它参与移植排斥反应、免疫炎症反应、非炎症性损伤等。本研究首次从haaliotis diversicolor中克隆了一个软体动物AIF-1基因HdAIF-1。其全长cDNA序列为942 bp,开放阅读框长度为456 bp,编码一个151 aa的蛋白。实时荧光定量PCR结果显示,HdAIF-1在所有组织中均有表达,其中以血淋巴和鳃中表达量最高。在温度胁迫下,HdAIF-1在温度升高期间在鳃中显著上调,在温度达到31℃时达到最高水平。而HdAIF-1在血淋巴和肝胰腺中的表达水平在热组与对照组之间从第1期到第4期均无显著差异,且在96 h时在这两个组织中的表达水平均显著上调。低氧应激下,血淋巴中HdAIF-1的表达水平在对照组和暴露组之间无显著差异。在鳃中,24 h显著下调,192 h显著上调。副溶血性弧菌攻毒后,HdAIF-1在血淋巴中的表达量在3 h达到最高水平,12 h降至对照水平,24 h和48 h均显著上调,提示HdAIF-1可能在不同应激条件下作为免疫因子发挥重要作用。
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引用次数: 1
Comparative analysis of genetic diversity of Dosidicus gigas from eastern Pacific Ocean based on molecular markers of mitochondrial DNA 基于线粒体DNA分子标记的东太平洋土sidicus gigas遗传多样性比较分析
Q4 Environmental Science Pub Date : 2013-01-01 DOI: 10.3724/SP.J.1231.2013.38490
L. Liu, Qianghua Xu, Xin-jun Chen, Jie Yan, Wei Yu, Cong-rong Wang
The worldwide climate and marine environmental changes have significant impacts on the distributions and fishery resources abundance fluctuations of Dosidicus gigas in the East Pacific Ocean. It is important to investigate genetic variance and population genetic structure of D. gigas in order to exploit and manage this squid scientifically. In this study,the genetic diversity and the genetic differentiation betw een tw o geographical populations of D. gigas w ere analyzed based on tw o mitochondrial DNA molecular markers. The results show ed that the number of haplotype,haplotype diversities,nucleotide diversities and average nucleotide differences w ere 7,( 0. 397 ± 0. 079),( 0. 001 09 ± 0. 000 96) and 0. 600 respectively in all Cytb sequences of tw o geographical populations,and haplotype diversity in population off the coast of Peru w as only( 0. 282 ± 0. 101) w hich w as significantly low er than that of equatorial w aters. On the other hand,the number of haplotype,haplotype diversities,nucleotide diversities and average nucleotide differences in all COⅠ sequences w ere 17,( 0. 787 ± 0. 051),( 0. 002 90 ± 0. 001 38) and 1. 802 respectively. The pairw ise fixation index F st and median netw ork revealed lack of genetic structure betw een populations of equatorial w aters and off the coast of Peru( Cytb: F st= 0. 055 23,P 0. 05; CO Ⅰ: F st= 0. 005 91,P 0. 05),and this might be caused by equational oceanographic current. The minimum spanning netw ork tree,mismatch distribution analysis and neutrality tests suggested that D. gigas experienced a recent population expansion,possibly 138 900 to 167 900 years ago.
全球气候和海洋环境变化对东太平洋大田螺的分布和渔业资源丰度波动有显著影响。研究巨乌贼的遗传变异和种群遗传结构,对科学开发和管理巨乌贼具有重要意义。本研究利用线粒体DNA分子标记分析了两个地理居群间的遗传多样性和遗传分化。结果表明,单倍型数量、单倍型多样性、核苷酸多样性和平均核苷酸差异分别为7、0。397±0。079),(0。001 09±0。000 96)和0。在2个地理种群的所有Cytb序列中,单倍型多样性分别为600个,秘鲁沿海种群的单倍型多样性仅为(0。282±0。101) w显著低于赤道w水域。另一方面,所有COⅠ序列的单倍型数量、单倍型多样性、核苷酸多样性和平均核苷酸差异w分别为17,(0。787±0。051),(0。002 90±0。001 38)和1。分别为802。配对固定指数F - 1和中位网络显示赤道w水域和秘鲁海岸种群之间缺乏遗传结构(Cytb: F - 1 = 0)。055 23, p 0。05;COⅠ:F st= 0。[05] 91, p . 0。05),这可能是由相等的海洋洋流引起的。最小跨越网络树、错配分布分析和中性测试表明,巨齿龙经历了最近的种群扩张,可能在13.89万年至16.79万年之间。
{"title":"Comparative analysis of genetic diversity of Dosidicus gigas from eastern Pacific Ocean based on molecular markers of mitochondrial DNA","authors":"L. Liu, Qianghua Xu, Xin-jun Chen, Jie Yan, Wei Yu, Cong-rong Wang","doi":"10.3724/SP.J.1231.2013.38490","DOIUrl":"https://doi.org/10.3724/SP.J.1231.2013.38490","url":null,"abstract":"The worldwide climate and marine environmental changes have significant impacts on the distributions and fishery resources abundance fluctuations of Dosidicus gigas in the East Pacific Ocean. It is important to investigate genetic variance and population genetic structure of D. gigas in order to exploit and manage this squid scientifically. In this study,the genetic diversity and the genetic differentiation betw een tw o geographical populations of D. gigas w ere analyzed based on tw o mitochondrial DNA molecular markers. The results show ed that the number of haplotype,haplotype diversities,nucleotide diversities and average nucleotide differences w ere 7,( 0. 397 ± 0. 079),( 0. 001 09 ± 0. 000 96) and 0. 600 respectively in all Cytb sequences of tw o geographical populations,and haplotype diversity in population off the coast of Peru w as only( 0. 282 ± 0. 101) w hich w as significantly low er than that of equatorial w aters. On the other hand,the number of haplotype,haplotype diversities,nucleotide diversities and average nucleotide differences in all COⅠ sequences w ere 17,( 0. 787 ± 0. 051),( 0. 002 90 ± 0. 001 38) and 1. 802 respectively. The pairw ise fixation index F st and median netw ork revealed lack of genetic structure betw een populations of equatorial w aters and off the coast of Peru( Cytb: F st= 0. 055 23,P 0. 05; CO Ⅰ: F st= 0. 005 91,P 0. 05),and this might be caused by equational oceanographic current. The minimum spanning netw ork tree,mismatch distribution analysis and neutrality tests suggested that D. gigas experienced a recent population expansion,possibly 138 900 to 167 900 years ago.","PeriodicalId":15710,"journal":{"name":"Journal of Fisheries of China","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2013-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"69967882","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Isolation of antibacterial peptides with low molecular weight from the spleen of Anguilla japonica 日本鳗鲡脾脏低分子量抗菌肽的分离
Q4 Environmental Science Pub Date : 2013-01-01 DOI: 10.3724/SP.J.1231.2013.38528
Ying-jiao Liang, R. Guan, Wen-shu Huang
The acetic acid extract of the spleen from Japanese eel,Anguilla japonica,was separated by tangential flow ultrafiltration with 10 and 3 ku fibre columns,and the two protein samples of the molecular weight(Mw)10 ku and the Mw3 ku were purified by using the cationic exchange liquid chromatography together with reverse-phase high-performance liquid chromatography(RP-HPLC).The results showed that three compositions(the protein samples of Mw10 ku,Mw10 ku and Mw3 ku)were separated from the acid extract with two fibre columns.The samples of Mw10 ku and Mw3 ku exhibited stronger antibacterial activity than the protein sample of the Mw10 ku.The protein sample with the Mw10 ku was purified by the cationic exchange liquid chromatography and using pH 9 buffer solution in RP-HPLC,and we obtained an antibacterial peptide with the molecular weight of 1 391.82 u when determined by MALDI-TOF MS.The pH 9 and the pH 2 buffer solutions were applied in RP-HPLC of the factions obtained from cationic exchange liquid chromatography of the protein sample with the Mw3 ku,and we also obtained an antibacterial peptide with the molecular weight of 839.19 u when determined by MALDI-TOF MS.This study suggests that the main antibacterial components in the spleen of A.japonica are the proteins of low molecular weight.
采用10和3 ku纤维柱切向流超滤分离日本鳗鲡脾脏乙酸提取物,并采用阳离子交换液相色谱和反相高效液相色谱(RP-HPLC)对分子量为10 ku和Mw3 ku的两种蛋白质样品进行纯化。结果表明,用两根纤维柱从酸提物中分离出了Mw10 ku、Mw10 ku和Mw3 ku蛋白样品。Mw10 ku和Mw3 ku蛋白样品的抑菌活性高于Mw10 ku蛋白样品。Mw10 ku的蛋白质样品由阳离子交换液相色谱纯化产物和使用9缓冲溶液pH值,我们获得一个抗菌肽的分子量1 391.82 u时由MALDI-TOF MS.The酸碱9和2缓冲派系获得的解决方案应用于rp -阳离子交换液相色谱的蛋白质样品Mw3 ku,我们也获得了抗菌肽的分子量MALDI-TOF ms测定值为839.19 u,提示粳稻脾脏中主要的抑菌成分为低分子量蛋白。
{"title":"Isolation of antibacterial peptides with low molecular weight from the spleen of Anguilla japonica","authors":"Ying-jiao Liang, R. Guan, Wen-shu Huang","doi":"10.3724/SP.J.1231.2013.38528","DOIUrl":"https://doi.org/10.3724/SP.J.1231.2013.38528","url":null,"abstract":"The acetic acid extract of the spleen from Japanese eel,Anguilla japonica,was separated by tangential flow ultrafiltration with 10 and 3 ku fibre columns,and the two protein samples of the molecular weight(Mw)10 ku and the Mw3 ku were purified by using the cationic exchange liquid chromatography together with reverse-phase high-performance liquid chromatography(RP-HPLC).The results showed that three compositions(the protein samples of Mw10 ku,Mw10 ku and Mw3 ku)were separated from the acid extract with two fibre columns.The samples of Mw10 ku and Mw3 ku exhibited stronger antibacterial activity than the protein sample of the Mw10 ku.The protein sample with the Mw10 ku was purified by the cationic exchange liquid chromatography and using pH 9 buffer solution in RP-HPLC,and we obtained an antibacterial peptide with the molecular weight of 1 391.82 u when determined by MALDI-TOF MS.The pH 9 and the pH 2 buffer solutions were applied in RP-HPLC of the factions obtained from cationic exchange liquid chromatography of the protein sample with the Mw3 ku,and we also obtained an antibacterial peptide with the molecular weight of 839.19 u when determined by MALDI-TOF MS.This study suggests that the main antibacterial components in the spleen of A.japonica are the proteins of low molecular weight.","PeriodicalId":15710,"journal":{"name":"Journal of Fisheries of China","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2013-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"69968008","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Cloning and expression analysis of farnesoic acid O-methyl transferase(FAMeT)gene during molting in Portunus trituberculatus 法尼松酸o -甲基转移酶(FAMeT)基因的克隆与表达分析
Q4 Environmental Science Pub Date : 2013-01-01 DOI: 10.3724/SP.J.1231.2013.38496
Xi Xie, Dongfa Zhu, Xiaoyu Cui, Jie Tang, Xier Qiu
To study the regulatory role of farnesoic acid O-methyl transferase(FAMeT)in molting process of crustaceans,the full-length FAMeT cDNA(GenBank accession number:KC192659)of Portunus trituberculatus is cloned by using reverse transcript PCR(RT-PCR)and rapid-amplification of cDNA ends(RACE).FAMeT cDNA contains a 201 bp 5′-untranslated region(5′-UTR),a 318 bp 3′-untranslated region(3′-UTR)and a 825 bp opening reading frame,which encodes 274 amino acid residues.Alignment of deduced amino acid sequence with FAMeT amino acid sequences of other crustaceans revealed that it shares the highest identity with P.pelagicus among the identities ranging from 75% to 97%.The amino acid residues consist of two copies of CF(CPAMD8/FAMeT)domain,which are the hallmark domain of FAMeT and are present in all crustacean FAMeTs.Quantitative real-time PCR(qRT-PCR)was used to quantify the relative expression level of FAMeT in different tissues and at molting stages in P.trituberculatus.The results showed that FAMeT was expressed in various tissues.The mRNA level of FAMeT was the highest in taoracic ganglia,followed by gill and mandibular organ(MO).During the molting process,the expression of FAMeT in MO increased to the maximum at D1 stage,then gradually decreased to the minimum at D4 stage.The results suggest that FAMeT plays an important role in molting regulation in P.trituberculatus.
为了研究法脂酸o -甲基转移酶(FAMeT)在甲壳类动物换壳过程中的调控作用,采用反转录PCR(RT-PCR)和cDNA末端快速扩增(RACE)技术克隆了三瘤猪(Portunus trituberculatus) FAMeT全长cDNA(GenBank登录号:KC192659)。FAMeT cDNA包含一个201 bp的5 ' -未翻译区(5 ' -UTR)、一个318 bp的3 ' -未翻译区(3 ' -UTR)和一个825 bp的开放阅读框,编码274个氨基酸残基。与其他甲壳类动物的FAMeT氨基酸序列比对表明,其与P.pelagicus的同源性最高,为75% ~ 97%。氨基酸残基由CF(CPAMD8/FAMeT)结构域的两个拷贝组成,这是FAMeT的标志结构域,存在于所有甲壳类动物的FAMeT中。采用实时荧光定量PCR(Quantitative real-time PCR, qRT-PCR)方法定量FAMeT在三瘤假体不同组织及蜕皮期的相对表达量,结果表明FAMeT在不同组织中均有表达。FAMeT mRNA的表达量以牛颈神经节最高,其次是鳃和下颌器官(MO)。在蜕皮过程中,MO中FAMeT的表达量在D1期最高,在D4期逐渐降低至最低。结果表明,FAMeT在三瘤棘鱼的蜕皮调控中起重要作用。
{"title":"Cloning and expression analysis of farnesoic acid O-methyl transferase(FAMeT)gene during molting in Portunus trituberculatus","authors":"Xi Xie, Dongfa Zhu, Xiaoyu Cui, Jie Tang, Xier Qiu","doi":"10.3724/SP.J.1231.2013.38496","DOIUrl":"https://doi.org/10.3724/SP.J.1231.2013.38496","url":null,"abstract":"To study the regulatory role of farnesoic acid O-methyl transferase(FAMeT)in molting process of crustaceans,the full-length FAMeT cDNA(GenBank accession number:KC192659)of Portunus trituberculatus is cloned by using reverse transcript PCR(RT-PCR)and rapid-amplification of cDNA ends(RACE).FAMeT cDNA contains a 201 bp 5′-untranslated region(5′-UTR),a 318 bp 3′-untranslated region(3′-UTR)and a 825 bp opening reading frame,which encodes 274 amino acid residues.Alignment of deduced amino acid sequence with FAMeT amino acid sequences of other crustaceans revealed that it shares the highest identity with P.pelagicus among the identities ranging from 75% to 97%.The amino acid residues consist of two copies of CF(CPAMD8/FAMeT)domain,which are the hallmark domain of FAMeT and are present in all crustacean FAMeTs.Quantitative real-time PCR(qRT-PCR)was used to quantify the relative expression level of FAMeT in different tissues and at molting stages in P.trituberculatus.The results showed that FAMeT was expressed in various tissues.The mRNA level of FAMeT was the highest in taoracic ganglia,followed by gill and mandibular organ(MO).During the molting process,the expression of FAMeT in MO increased to the maximum at D1 stage,then gradually decreased to the minimum at D4 stage.The results suggest that FAMeT plays an important role in molting regulation in P.trituberculatus.","PeriodicalId":15710,"journal":{"name":"Journal of Fisheries of China","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2013-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"69968017","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 3
Effects of the toxic dinoflagellate Alexandrium tamarense on MDA,SOD and GST in hepatopancreas and gill of Fenneropenaeus chinensis 有毒鞭毛藻tamarense Alexandrium对中国对虾肝胰腺和鳃中MDA、SOD和GST的影响
Q4 Environmental Science Pub Date : 2013-01-01 DOI: 10.3724/SP.J.1231.2013.38501
Zhongxiu Liang, Li Jian, Zhijun Tan, Jitao Li, Junping Liang, Hongxing Ge
This study probes into effects of the toxic dinoflagellate Alexandrium tamarense(ATHK),a producer of paralytic shellfish poison,on antioxidant system of Chinese shrimp Fenneropenaeus chinensis,an important mariculture species in China.The shrimp were intramuscularly injected the crude toxin extracted from A.tamarense cells.The dose injection was carried out only one time during the experiment,using extracted solution from 1.4×103 algae cells.Superoxide dismutase(SOD)activity,glutathione-S-transferase(GST)activity and malondialdehyde(MDA)content were analyzed in hepatopancreas and gill at 1,3,6,12,24 and 48 h after stress.SOD activity and GST activity in hepatopancreas and gill increased within 6 h.However,they were inhibited in gill at 12 h and 48 h after stress.MDA content in hepatopancreas had no significant change except at 1 h after stress,but increased in gill with time prolonging.The results indicated that the crude toxin extracted from A.tamarense cells could cause lipid peroxidation in gill of F.chinensis by induction of MDA increase and SOD and GST inactivation.
本研究探讨了产生麻痹性贝类毒素的有毒甲藻亚历山大藻(Alexandrium tamarense, ATHK)对中国重要海水养殖品种中国对虾(Fenneropenaeus chinensis)抗氧化系统的影响。将从塔玛氏A.tamarense细胞中提取的粗毒素肌内注射。实验过程中只进行一次剂量注射,使用1.4×103藻类细胞提取液。分别于应激后1、3、6、12、24和48 h测定肝胰腺和鳃超氧化物歧化酶(SOD)活性、谷胱甘肽- s转移酶(GST)活性和丙二醛(MDA)含量。肝胰脏和鳃中SOD活性和GST活性在应激后6 h内升高,但在应激后12 h和48 h,鳃中SOD活性和GST活性均受到抑制。除应激1 h外,肝胰脏MDA含量无显著变化,但随着应激时间的延长,肝胰脏MDA含量呈上升趋势。结果表明,粗毒素可诱导中华绒螯虾鳃脂质过氧化,使其MDA升高,SOD和GST失活。
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引用次数: 0
Acute effects of ammonia exposure on selected immunological and metabolic parameters in juvenile red claw crayfish(Cherax quadricarinatus)and the post-exposure recovery 氨暴露对红爪螯虾幼虾特定免疫代谢参数的急性影响及暴露后恢复
Q4 Environmental Science Pub Date : 2013-01-01 DOI: 10.3724/SP.J.1231.2013.38563
Jiang Qichen, Gui Shuyu, Zhang Wenyi, Z. Chengxiang, Feng Xiaoqing, Tan HongYue, Huang Wenting, Yang Jiaxin, Li Feng
The acute effects of ammonia exposure on selected immunological and metabolic parameters in juvenile red claw crayfish,Cherax quadricarinatus as well as the post-exposure recovery were studied by using Real-time PCR technique and biochemical assays.The crayfish were first exposed to ammonia for 3 d,then half of them were sampled and the other half were moved into aerated tap water for a 7 d post-exposure recovery.The results showed that a 3 d ammonia exposure had significant impact on the activities of ACP,AKP and SOD in the muscle of juvenile C.quadricarinatus.As ammonia concentration increased,the activities of these enzymes significantly decreased.At the highest concentration(16 mg/L),they decreased to 76%,68% and 62% of the control,respectively.The mRNA expression of mitochondrial manganese superoxide dismutase(mMnSOD),cytosolic MnSOD(cMnSOD),extracellular copper/zinc SOD(exCu/ZnSOD)also showed a decreasing trend following increased ammonia concentration.In contrast,the mRNA expression of catalase(CAT),glutathione peroxidase(GPX)and glutathione transferase(GST)in muscle remained unchanged compared to the control.Dissolved protein and triglycerides in hepatopancreas significantly decreased as ammonia concentration increased.At the highest concentration,they decreased to 72% and 59% of the control.AST showed a noble elevation which was 134% of the control in 12 mg/L group.After a 7 d post-exposure recovery,ACP and AKP activities returned to the normal level in ammonia treatments.On the contrary,the activities of SOD and GPX in all ammonia treatments were significantly higher than the control.The expression of antioxidant genes was higher than the control in all ammonia treatments.Metabolic parameters showed no differences between all groups.The results indicate that high levels of ammonia might suppress activities and gene expression patterns of immune-related enzymes,leading to a loss of defence mechanism.The crayfish which had been exposed to ammonia for 3 d could not fully recover after a 7 d post-exposure recovery and oxidative stress still existed in the muscle in C.quadricarinatus.
采用Real-time PCR技术和生化检测方法,研究了氨暴露对红爪小龙虾幼虾部分免疫代谢指标及暴露后恢复的急性影响。首先将小龙虾暴露于氨中3 d,然后将其中一半取样,另一半放入加气的自来水中进行暴露后7 d的恢复。结果表明,3 d氨处理对黄鳝幼鱼肌肉中ACP、AKP和SOD活性有显著影响,且随着氨处理浓度的升高,这些酶的活性显著降低。在最高浓度(16 mg/L)时,它们分别下降到对照的76%、68%和62%。线粒体锰超氧化物歧化酶(mMnSOD)、胞质锰超氧化物歧化酶(cMnSOD)、细胞外铜/锌超氧化物歧化酶(exCu/ZnSOD) mRNA表达量也随氨浓度的升高呈下降趋势。与对照组相比,肌肉中过氧化氢酶(CAT)、谷胱甘肽过氧化物酶(GPX)和谷胱甘肽转移酶(GST) mRNA表达量保持不变。肝胰脏溶解蛋白和甘油三酯随着氨浓度的升高而显著降低。在最高浓度时,它们分别下降到对照的72%和59%。12 mg/L组AST显著升高,为对照组的134%。暴露后恢复7 d后,氨处理的ACP和AKP活性恢复到正常水平。相反,各氨处理的SOD和GPX活性均显著高于对照。各氨处理抗氧化基因的表达量均高于对照。各组间代谢参数无显著差异。结果表明,高水平的氨可能抑制免疫相关酶的活性和基因表达模式,导致防御机制丧失。暴露于氨3 d后的小龙虾在7 d后仍不能完全恢复,肌肉中仍存在氧化应激。
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引用次数: 1
Prokaryotic expression of C-type lectin gene of Litopenaeus vannamei and immunologic activity evaluation of recombinant protein 凡纳滨对虾c型凝集素基因的原核表达及重组蛋白的免疫活性评价
Q4 Environmental Science Pub Date : 2013-01-01 DOI: 10.3724/SP.J.1231.2013.38572
Jinhong Yu, L. Pan, Hui Zhang
The studies on innate immune responses of Litopenaeus vannamei C-type lectin,were conducted by recombinant C-type lectin protein through expression in Escherichia coli.We used cell suspension culture to study the effect of C-type lectin on the cellular immune response of Litopenaeus vannamei.The experiment was divided into two groups:control group and recombinant protein(1.0 mg/mL).After 0,3,6,9,12,24 hours,we harvested the solutions and then assessed parameters respectively.The results showed that SDS-PAGE revealed the molecular weight of LvLec protein was 39 ku,which was consistent with the predicted size.Western-blotting revealed it could bind anti-His antibody,which proved that C-type lectin recombinant protein in Escherichia coli succeeded in expression.For the hemagglutination assay,massive agglutination could be observed after Escherichia coli was incubated with LvLec for 1 h.At the same time,LvLec protein had a significant effect on haemocyte count,cell viability and phagocytic activity of white shrimp Litopenaeus vannamei(P0.05).The extracelluar phenoloxidase activity of the LvLec protein group showed peak changes during 3-12 h,and at 9 h the extracelluar phenoloxidase activity was highest,then returned to the control level(P0.05).Therefore,crustacean C-type lectin not only had the function of hemagglutination,but also activated prophenoloxidase activating system,and probably played an important role in immune response.
通过重组c型凝集素蛋白在大肠杆菌中的表达,对凡纳滨对虾c型凝集素的先天免疫应答进行了研究。采用细胞悬浮培养法研究c型凝集素对凡纳滨对虾细胞免疫应答的影响。实验分为两组:对照组和重组蛋白(1.0 mg/mL)。在0、3、6、9、12、24小时后,我们分别收集溶液并评估参数。结果显示,SDS-PAGE显示LvLec蛋白的分子量为39 ku,与预测的大小一致。Western-blotting结果显示,该蛋白可结合抗his抗体,证明c型凝集素重组蛋白在大肠杆菌中表达成功。在血凝试验中,LvLec与大肠杆菌孵育1 h后可观察到大量的血凝现象,同时LvLec蛋白对凡纳滨对虾的血细胞计数、细胞活力和吞噬活性均有显著影响(P0.05)。LvLec蛋白组胞外酚氧化酶活性在3 ~ 12 h变化最大,在9 h达到最高值,之后恢复到对照水平(P0.05)。因此,甲壳类动物c型凝集素不仅具有血凝功能,还能激活酚氧化酶原激活系统,可能在免疫应答中发挥重要作用。
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