Heritable targeted inactivation of myostatin gene in yellow catfish (Pelteobagrus fulvidraco) using engineered zinc finger nucleases

Yellow catfish (Pelteobagrus fulvidraco) is one of the most important freshwater aquaculture species in China. However, its small size and lower meat yield limit its edible value. Myostatin (MSTN) is a negative regulator of mammalian muscle growth. But, the function of Mstn in fish remains elusive. To explore roles of mstn gene in fish growth and create a strain of yellow catfish with high amount of muscle mass, we performed targeted disruption of mstn in yellow catfish using engineered zinc-finger nucleases (ZFNs). Employing zebrafish embryos as a screening system to identify ZFN activity, we obtained one pair of ZFNs that can edit mstn in yellow catfish genome. Using the ZFNs, we successfully obtained two founders (Founder July29-7 and Founder July29-8) carrying mutated mstn gene in their germ cells. The mutated mstn allele inherited from Founder July29-7 was a null allele (mstn(nju6)) containing a 4 bp insertion, predicted to encode function null Mstn. The mutated mstn inherited from Founder July29-8 was a complex type of mutation (mstn(nju7)), predicted to encode a protein lacking two amino acids in the N-terminal secretory signal of Mstn. Totally, we obtained 6 mstn(nju6/+) and 14 mstn(nju7/+) yellow catfish. To our best knowledge, this is the first endogenous gene knockout in aquaculture fish. Our result will help in understanding the roles of mstn gene in fish.     

黄颡鱼MHC class II基因全长的克隆及饲料维生素D3对其组织表达的影响

为进一步丰富鱼类MHC class II基因的研究, 同时也为进一步探讨低磷饲料中添加维生素D₃对鱼类免疫功能可能的影响, 实验利用RACE (Rapid-amplification of cDNA ends) 即cDNA末端快速扩增技术, 成功克隆出黄颡鱼(Pelteobagrus fulvidraco)主要组织相容性复合体(Major histocompatibility complex, MHC) class II基因, 全长1074 bp, 其中ORF (Open reading frame)708 bp, 编码236个氨基酸, 5′UTR (5′端非翻译区)78 bp, 3′UTR (3′端非翻译区)259 bp。进行氨基酸序列比对分析得到: 黄颡鱼MHC class II基因ORF氨基酸序列与长吻逘(Leiocassis longirostris)的氨基酸序列相似度最高为69.5%, 与锦鲤(Cyprinus carpio)的氨基酸序列相似度最低为50.4%。利用qPCR对黄颡鱼MHC class II基因进行组织表达分析, 结果表明MHC class II在小肠、肝脏、鳃中表达较高; 在肌肉、鳍条中表达较低; 而在肾、脾脏、脑、头肾中表达量极低(几乎检测不到)。在低磷饲料中添加维生素D₃显著诱导了该基因的上调表达。研究结果展示了黄颡鱼MHC class II基因的分子结构、组织表达以及维生素D₃的作用, 在降低磷排放的同时, 为今后黄颡鱼免疫抗病及分子选育等方向的深入研究及免疫型饲料的使用奠定了基础。     

Isolation of yellow catfish β-actin promoter and generation of transgenic yellow catfish expressing enhanced yellow fluorescent protein

Yellow catfish (Pelteobagrus fulvidraco Richardson) is one of the most important freshwater farmed species in China. However, its small size and slow growth rate limit its commercial value. Because genetic engineering has been a powerful tool to develop and improve fish traits for aquaculture, we performed transgenic research on yellow catfish in order to increase its size and growth rate. Performing PCR with degenerate primers, we cloned a genomic fragment comprising 5'-flanking sequence upstream of the initiation codon of β-actin gene in yellow catfish. The sequence is 1,017?bp long, containing the core sequence of proximal promoter including CAAT box, CArG motif and TATA box. Microinjecting the transgene construct Tg(beta-actin:eYFP) of the proximal promoter fused to enhanced yellow fluorescent protein (eYFP) reporter gene into zebrafish and yellow catfish embryos, we found the promoter could drive the reporter to express transiently in both embryos at early development. Screening the offspring of five transgenic zebrafish founders developed from the embryos microinjected with Tg(ycbeta-actin:mCherry) or 19 yellow catfish founders developed from the embryos microinjected with Tg(beta-actin:eYFP), we obtained three lines of transgenic zebrafish and one transgenic yellow catfish, respectively. Analyzing the expression patterns of the reporter genes in transgenic zebrafish (Tg(ycbeta-actin:mCherry)nju8/+) and transgenic yellow catfish (Tg(beta-actin:eYFP)nju11/+), we found the reporters were broadly expressed in both animals. In summary, we have established a platform to make transgenic yellow catfish using the proximal promoter of its own β-actin gene. The results will help us to create transgenic yellow catfish using "all yellow catfish" transgene constructs.     

Overexpression of Myostatin2 in zebrafish reduces the expression of dystrophin associated protein complex (DAPC) which leads to muscle dystrophy

Myostatin, a member of the TGF-β superfamily, is a potent negative regulator of skeletal muscle and growth. Previously, we reported Mstn1 from zebrafish and studied its influence on muscle development. In this study, we identified another form of Myostatin protein which is referred to as Mstn2. The size of Mstn2 cDNA is 1342 bp with 109 and 132 bp of 5′ and 3′-untranslated regions (UTRs), respectively. The coding region is 1101 bp encoding 367 amino acids. The identity between zebrafish Mstn1 and 2 is 66%. The phylogenetic tree revealed that the Mstn2 is an ancestral form of Mstn1. To study the functional aspects, we overexpressed mstn2 and noticed that embryos became less active and the juveniles with bent and curved phenotypes when compared to the control. The RT-PCR and in situ hybridization showed concurrent reduction of dystrophin associated protein complex (DAPC). In cryosection and in situ hybridization, we observed the disintegration of somites, lack of transverse myoseptum and loss of muscle integrity due to the failure of muscle attachment in mstn2 overexpressed embryos. Immunohistochemistry and western blot showed that there was a reduction of dystrophin, dystroglycan and sarcoglycan at translational level in overexpressed embryos. Taken together, these results indicate the suitability of zebrafish as an excellent animal model and our data provide the first in vivo evidence of muscle attachment failure by the overexpression of mstn2 and it leads to muscle loss which results in muscle dystrophy that may contribute to Duchenne syndrome and other muscle related diseases. A. Anusha Amali and Cliff Ji-Fan Lin contributed equally.     

Urokinase is required for the formation of mactinin,an alpha-actinin fragment that promotes monocyte/macrophage maturation

Myostatin is a recently discovered gene that inhibits muscle growth. In the present study, we characterized the myostatin locus and its expression in channel catfish (Ictalurus punctatus). The genomic DNA and cDNA encoding the channel catfish myostatin were cloned and sequenced. The myostatin gene has three exons encoding a protein of 389 amino acids. Comparison of the genomic sequences with those of the cDNA revealed that the myostatin cDNA was 1673 base pair (bp) long with a 5'-untranslated region (UTR) and 3'-UTR of 180 and 323 bp, respectively. The deduced amino acid sequences of the catfish myostatin is highly conserved with those of other organisms. The myostatin locus is highly polymorphic in channel catfish because of the presence of several microsatellites and single nucleotide polymorphic sites. The myostatin gene was expressed in various tissues and developmental stages at differential levels, suggesting complex regulation of this gene and perhaps roles for myostatin in addition to those originally suggested.     

低蛋白饲料对杂交黄颡鱼幼鱼生长性能、体组成、转氨酶活性和抗氧化能力的影响

本实验以杂交黄颡鱼(Pelteobagrus fulvidraco♀ ×Pelteobagrus vachelli♂)幼鱼为研究对象,探究低蛋白饲料中补充蛋氨酸和赖氨酸对杂交黄颡鱼的生长性能、体组成、转氨酶活性及抗氧化能力的影响.实验设计5组饲料,设置3个蛋白梯度(42％、37％和32％),分别记为对照组、LP1组和L...     

黄颡鱼HSC70基因及其组织表达分析

热休克蛋白70（HSP70）与生物体的抗胁迫能力密切相关。本文采用RACE (Rapid amplification of cDNA ends) 技术,从黄颡鱼Pelteobagrus fulvidraco克隆到一种组成型热休克蛋白（HSC70）基因及其cDNA。该cDNA全长2245bp,包括5′非编码区82bp,3′非编码区225bp,开放阅读框(ORF) 1938bp,编码645个氨基酸组成的蛋白质。黄颡鱼HSC70基因含有8个内含子,与人、鼠、虹鳟和花斑溪鳉的HSC70基因内含子数目相同,位置相似。其中,最长内含子（873bp）位于5′端非编码区,其余内含子（长度在80－251bp之间不等）均在编码区以内。黄颡鱼HSC70基因编码的氨基酸序列与南方鲶的相似度最高,达96.13%,与欧洲银鲫和团头鲂的相似度分别为94.45%和94.14%。RT-PCR检测显示,正常情况下黄颡鱼HSC70在血细胞、心脏、肝、头肾、脾、鳃、肌肉和脑中均有表达,但表达量在鳃中最高,肌肉中最低;统计结果显示,热激后HSC70在血细胞、肝、头肾和脑中的表达量显著上升(p<0.05),而在其余组织中热激前后的表达差异不显著(p>0.05)。     

Interferon-alpha genes from Bos and Bubalus bubalus

Interferon-a genes were cloned from six breeds of three species of two genera (three Chinese native cattle breeds of yellow cattle, wild yak and HuanHu domestic yak, one European breed of Holstein cow, and two water buffalo breeds of FuAn water buffalo and FuZhong water buffalo) by direct PCR. The PCR products were directly inserted into the expression vector to be sequenced and expressed. Sequence analysis showed that IFN-a genes of six clones were composed of 498 nucleotides, encoding a mature polypeptide with 166 amino acids. Compared with the published BoIFN-a subtypes, the IFN-a gene of Holstein cow had only one point mutation with the BoIFN-aA subtype. The IFN-a gene of yellow cattle was similar to the BoIFN-aD subtype with amino acid identity of 97.0% and may be considered as a new subtype, namely, BoIFN-aD1. The other four IFN-a genes, cloned from wild yak and HuanHu domestic yak, FuAn water buffalo, and FuZhong water buffalo, represented four new subtypes, namely, BoIFN-aI, BoIFN-aJ, BuIFN-a1, and BuIFN-a2, respectively. Each of the six clones was expressed in E. coli with molecular weight of approximately 20 kDa by SDS-PAGE and Western blot analyses. Antiviral activity assays showed that the six recombinant IFN-a (rIFN-a) all exhibited 1,000 times higher antiviral activity in the MDBK/VSV cell line than in the CEF/VSV one. Moreover, the rIFN-as could inhibit infectious bovine rhinotracheitis virus replication in the MDBK cell line using CPE inhibition method. The results suggested that rIFN-as a potential agent for clinical application against virus diseases in cattle industry.     

重组鲁西黄牛α干扰素融合蛋白的表达及其抗病毒活性研究

通过PCR从鲁西黄牛(Yellowcattle)基因组DNA中克隆了α干扰素(BoIFN-α)基因,并插入到pET32a 中,构建成重组原核表达质粒pET32a /BoIFN-α,进行测序和诱导表达。测序结果表明,鲁西黄牛IFN-α基因全长498个核苷酸,含一个开放阅读框(ORF),编码166个氨基酸的成熟蛋白,与已报道的牛α干扰素C亚型氨基酸组成同源性为97.6%。表达产物经SDS-PAGE分析,表达出40kD的融合蛋白,表达量占菌体总蛋白的26.7%。表达产物经镍离子螯合次氨基三乙酸(Ni-NTA)亲和层析纯化,纯化产物进行复性后在MDBK/VSV上的活性为5×105u/mg。重组牛IFN-α(rBoIFN-α)对牛轮状病毒(BRV)有一定的抑制作用,抗BRV病毒活性为1.5×105u/mg。结果显示从鲁西黄牛中克隆了IFN-α基因的一种新亚型,即BoIFN-αC2,并实现了高效表达,获得了具有较高抗病毒活性的重组干扰素产物,为重组牛干扰素的开发奠定了基础。     

Cloning and expression analysis of c-type and g-type lysozymes in yellow catfish (<Emphasis Type="Italic">Pelteobagrus fulvidraco</Emphasis>)

Lysozymes have important roles in innate immune system. Here, a c-type and a g-type lysozyme were identified from yellow catfish (Pelteobagrus fulvidraco). The deduced amino acid sequences of both lysozymes were conserved in catalytic sites and structural features as compared to their counterparts from other species. It was interesting that the g-type lysozyme possessed a signal peptide. The c-type and g-type lysozymes had the highest identity 89.4 and 76.2 % with that from channel catfish respectively. Phylogenetic analysis showed that the two lysozymes had a closely relationship with that from channel catfish and Astyanax mexicanus. Lysozymes from one order could form more than one clade in the phylogenetic tree, which indicated the gene duplications in evolution. Expression analysis with real time quantitative PCR revealed that the two lysozyme genes were constitutively expressed in all the tested tissues. The highest expression of c-type lysozyme was observed in liver, followed by spleen, head kidney, and trunk kidney, while the g-type lysozyme had highest expression in intestine, followed by spleen, head kidney, and trunk kidney. The mRNA levels of both genes were all up-regulated after challenging with Aeromonas hydrophila. However, there were differences in tissues and time points when the mRNA levels reached its peak between the two lysozymes. It indicated the diversity in regulation mechanisms and detailed functions among lysozymes. Taking together, these results will benefit the understanding of yellow catfish lysozymes.     

杂交黄颡鱼与普通黄颡鱼幼鱼生长性能及耐低氧能力的比较

实验以杂交黄颡鱼(Pelteobagrus fulvidraco)和普通黄颡鱼幼鱼为实验对象, 拟通过8周的投喂生长和低氧胁迫实验, 比较研究杂交黄颡鱼与普通黄颡鱼的生长性能及耐低氧抗逆性。投喂生长实验: 经过8周的养殖, 杂交黄颡鱼平均体重为(19.60±0.88) g/尾, 显著高于普通黄颡鱼平均体重为(15.74±0.42) g/尾(P<0.05), 杂交黄颡鱼幼鱼较普通黄颡鱼幼鱼体重生长快24.52%; 杂交黄颡鱼幼鱼存活率为(87.78±1.92)%, 显著高于普通黄颡鱼幼鱼存活率(67.78±1.92)% (P<0.05), 杂交黄颡鱼幼鱼比普通黄颡鱼幼鱼存活率高 29.51%; 杂交黄颡鱼的饲料系数为1.18±0.14, 普通黄颡鱼饲料系数为1.36±0.21。低氧胁迫实验: 同时将杂交黄颡鱼和普通黄颡鱼置于在溶氧量(1.48 ± 0.27) mg/L的水体中, 分别在低氧胁迫0、6h、12h和24h后, 检测血清和肝脏中抗氧化酶活性以及脑和肝脏中缺氧诱导基因(HIF-1α)的相对表达量发现: 杂交黄颡鱼和普通黄颡鱼血清和肝脏中乳酸脱氢酶(LDH)活性、超氧化物歧化酶(SOD)活性和过氧化氢酶(CAT)活性在低氧胁迫后 6h以及总抗氧化能力(T-AOC)在低氧胁迫后 12h较低氧胁迫 0均出现显著性变化(P<0.05)且在低氧胁迫6h、12h和24h杂交黄颡鱼抗氧化酶活性均高于普通黄颡鱼; 杂交黄颡鱼和普通黄颡鱼脑和肝脏中缺氧诱导基因(HIF-1α)的相对表达量均在低氧胁迫后出现显著性上升(P<0.05)且在低氧胁迫6h、12h和 24h杂交黄颡鱼缺氧诱导基因(HIF-1α)的相对表达量均高于普通黄颡鱼。从无氧代谢能力、抗氧化能力以及缺氧诱导基因相对表达量3方面分析表明杂交黄颡鱼和黄颡鱼低氧胁迫短时间均具有一定的低氧耐受能力但随着胁迫时间延长均会出现氧化损伤且杂交黄颡鱼的耐低氧能力要显著性高于普通黄颡鱼。     

Cloning and characterisation of a channel catfish (Ictalurus punctatus) Mx gene

A 2.5 kb full-length cDNA clone of a channel catfish (Ictalurus punctatus) Mx gene was obtained using RACE (rapid amplification of cDNA ends) polymerase chain reaction (PCR) from RNA extracted from the liver of poly I:C stimulated channel catfish. The gene consists of an open reading frame of 1905 nucleotides encoding a 635 amino acid protein. The predicted protein is 72.5 kDa and contains the dynamin family signature, a tripartite GTP binding motif and a leucine zipper, characteristic of all known Mx proteins. The catfish Mx protein exhibited 79% identity with perch Mx and between 71% and 74% identity with the three Atlantic salmon and the three rainbow trout Mx proteins. Mx mRNA was constitutively expressed in channel catfish ovary (CCO) cells, but in higher quantities in response to poly I:C treatment. Mx was induced in channel catfish following injection with channel catfish virus (CCV) and poly I:C.     

Cloning and functional characterization of a gonadal luteinizing hormone receptor complementary DNA from the African catfish (Clarias gariepinus)

A cDNA encoding a putative African catfish (Clarias gariepinus) gonadal LH receptor (cfLH-R) has been cloned. Multiple sequence alignment of the deduced amino acid sequence revealed that the cfLH-R had the highest identity with vertebrate LH receptors (>50%). Overall sequence identity between the cfLH-R and the African catfish FSH receptor (cfFSH-R) is 47%. Sequence analysis of part of the cfLH-R gene revealed the presence of an intron typically found in other vertebrate LH-R genes. Abundant cfLH-R mRNA expression was detected in ovary and testis as well as in head-kidney (the adrenal homologue in fish). Other tissues, such as muscle, brain, cerebellum, stomach, heart, and seminal vesicles, also contained detectable cfLH-R mRNA. Transient expression of the cfLH-R in HEK-T 293 cells resulted in significantly increased basal cAMP levels in the absence of gonadotropic hormone. The cAMP levels could be further elevated in response to catfish LH, salmon LH, human LH, human choriogonadotropin, and human FSH. Salmon FSH and human TSH, however, were inactive. We conclude that we have cloned a cDNA encoding the LH-R of the African catfish. This receptor displays constitutive activity but is still responsive to additional ligand-induced activation.     

Interferon-α Genes from Bos and Bubalus bubalus

Interferon-α genes were cloned from six breeds of three species of two genera (three Chinese native cattle breeds of yellow cattle, wild yak and HuanHu domestic yak, one European breed of Holstein cow, and two water buffalo breeds of FuAn water buffalo and FuZhong water buffalo) by direct PCR. The PCR products were directly inserted into the expression vector to be sequenced and expressed. Sequence analysis showed that IFN-α genes of six clones were composed of 498 nucleotides, encoding a mature polypeptide with 166 amino acids. Compared with the published BoIFN-α subtypes, the IFN-α gene of Holstein cow had only one point mutation with the BoIFN-αA subtype. The IFN-α gene of yellow cattle was similar to the BoIFN-αD subtype with amino acid identity of 97.0% and may be considered as a new subtype, namely, BoIFN-αD1. The other four IFN-α genes, cloned from wild yak and HuanHu domestic yak, FuAn water buffalo, and FuZhong water buffalo, represented four new subtypes, namely, BoIFN-αI, BoIFN-αJ, BuIFN-α1, and BuIFN-α2, respectively. Each of the six clones was expressed in E. coli with molecular weight of ～ 20kDa by SDS-PAGE and Western blot analyses. Antiviral activity assays showed that the six recombinant IFN-α (rIFN-α) all exhibited 1000 times higher antiviral activity in the MDBK/VSV cell line than in the CEF/VSV one. Moreover, the rIFN-αs could inhibit infectious bovine rhinotracheitis virus replication in the MDBK cell line using CPE inhibition method. The results suggested that rIFN-αs a potential agent for clinical application against virus diseases in cattle industry.     

肌肉增强子因子2对猪肌肉生长抑制素启动子活性的调节

肌肉生长抑制素(Myostatin,Mstn)是转化生长因子-β超家族的成员,在哺乳动物的骨骼肌生长和分化过程中起负调控作用,其转录调控受到多个基因的影响,其中肌肉增强子因子2(MEF2)是重要的调控因子之一。因此,对猪Mstn启动子上MEF2位点及其作用方式的探讨具有重要意义。首先,通过PCR方法扩增了猪Mstn基因上游1 969 kb的启动子序列,利用生物信息学方法分析该序列包含3个MEF2的结合位点;其次,采用逐步删除的方法获得5个长度不等的启动子,用荧光素酶报告系统评估了它们在小鼠成肌细胞C2C12中的活性。其次,转入含有MEF2结合位点的启动子片段和MEF2C表达载体,可以显著增强启动子活性2～6倍,高表达另一亚型MEF2A则启动子活性没有明显改变。最后,转入MEF2C表达载体,用实时定量PCR和Western blotting方法检测Mstn的转录和蛋白水平的变化,结果发现mRNA升高了2～4倍;在肌管细胞中,蛋白翻译水平也有显著升高。这些结果显示,MEF2C可以通过激活Mstn参与猪肌肉生长和发育阶段的调节。研究为Mstn基因的转录调控提供了有效的作用靶点和效应分子,为进一步探讨Mstn的功能调控提供了一种新的思路。     

抗菌肽HBβ-C在全雄和杂交黄颡鱼对多子小瓜虫抗性差异中的作用

为研究全雄黄颡鱼(Pelteobagrus fulvidraco)、瓦式黄颡鱼(Pelteobagrus vachelli)和杂交黄颡鱼(黄颡鱼P.fulvidraco♀×瓦氏黄颡鱼P. vachelli♂)对多子小瓜虫(Ichthyophthirius multifiliis)的抗性差异,通过生物信息学分析黄颡鱼皮肤黏液蛋白质组,发现其血红蛋白源抗菌肽(HBβ-C)位于血红蛋白β链HBβ的碳端,共33个氨基酸。利用化学合成的不同浓度的HBβ-C肽段进行体外抗虫实验,研究发现其能有效杀死滋养体、包囊体和掠食体阶段的多子小瓜虫,其中15μg/mL的HBβ-C能在3min内杀死所有滋养体。基因表达量分析显示,在杂交黄颡鱼的鳃和皮肤组织中, HBβ的mRNA表达量高于全雄黄颡鱼;但在应对小瓜虫感染的过程中,全雄黄颡鱼的HBβmRNA转录水平快速提升,其表达水平和上升倍率显著高于杂交黄颡鱼。蛋白表达量分析显示,HBβ在全雄黄颡鱼鳃组织中的蛋白表达量明显高于杂交黄颡鱼。免疫荧光定位结果显示,抗菌肽HBβ-C特异地在红细胞中表达,可以分泌并附着在滋养体上。综上所述,相对于杂交黄颡鱼,全雄黄颡鱼中H...     

Chemical identification of catfish growth hormone and prolactin.

Isolation and primary structure of growth hormone (GH) and prolactin (PRL) from the pituitary gland of catfish (Ictalurus punctatus) are described. Alkaline extract of the pituitary glands was fractionated by gel filtration on Sephadex G-75, ion-exchange chromatography on DEAE-cellulose, and reversed-phase high-performance liquid chromatography on Octadecyl silica ODS. Catfish GH and PRL were identified by Western blotting with antisera against chum salmon GH and PRL. The catfish GH consists of 178 residues and is the most similar to carp GH, with sequence identity of 77%, although there is an uninterrupted deletion of 10 amino acid residues that corresponds to carp GH (90-99). The PRL is composed of 187 residues, which also exhibits the highest identity (79%) with carp PRL. Sequence identity between catfish GH and PRL is only 27%.     

Muscle growth after postdevelopmental myostatin gene knockout

Constitutive myostatin gene knockout in mice causes excessive muscle growth during development. To examine the effect of knocking out the myostatin gene after muscle has matured, we generated mice in which myostatin exon 3 was flanked by loxP sequences (Mstn[f/f]) and crossed them with mice bearing a tamoxifen-inducible, ubiquitously expressed Cre recombinase transgene. At 4 mo of age, Mstn[f/f]/Cre+ mice that had not received tamoxifen had a 50-90% reduction in myostatin expression due to basal Cre activity but were not hypermuscular relative to Mstn[w/w]/Cre+ mice (homozygous for wild-type myostatin gene). Three months after tamoxifen treatment (initiated at 4 mo of age), muscle mass had not changed from the pretreatment level in Mstn[w/w]/Cre+ control mice. Tamoxifen administration to 4-mo-old Mstn[f/f]/Cre+ mice reduced myostatin mRNA expression to less than 1% of normal, which increased muscle mass approximately 25% over the following 3 mo in both male and female mice (P<0.005 vs. control). Fiber hypertrophy appeared to be sufficient to explain the increase in muscle mass. The pattern of expression of genes encoding the various myosin heavy-chain isoforms was unaffected by postdevelopmental myostatin knockout. We conclude that, even after developmental muscle growth has ceased, knockout of the myostatin gene induces a significant increase in muscle mass.     

番木瓜环斑病毒畸叶株系的CP基因克隆和序列分析

采用RT－PCR方法合成了本研究室保存的番木瓜畸叶病毒（PMaLV）的外壳蛋白（CP）基因,将其CP基因克隆进Promega公司的pGEM-T and pGEM-T Easy Vector System（简称T－载体）,并进行了序列分析。结果表明,PMaLV CP基因核苷酸序列全长为861nt,推导其编码287个氨基酸。与番木瓜环斑病毒（PRSV）美国夏威HA株系和澳大利亚W株系的CP基因相比,在第66nt处开始连续缺失3个核苷酸。与PRSV的华南Ys、Sm和G株系以及夏威夷的HA和澳大利亚的W株系相比,其CP基因序列同源率分别为96％、98％、95％、89％和89％。其的氨基酸序列同源率分别为98％、97％、97％、96％和95％。此结果表明,PMaLV属于PRSV的一个株系,不是一种新病毒。因此,我们称其为番木瓜环斑病毒畸叶株系（ML株系）。