高羊茅叶绿体表达载体构建及绿色荧光蛋白基因瞬时表达检测

 【目的】验证高羊茅叶绿体表达载体在高羊茅叶绿体中的瞬时表达情况,为今后在高羊茅叶绿体中稳定表达该载体,获得耐旱高羊茅的叶绿体转基因株系奠定基础。【方法】首先从高羊茅草叶绿体基因组中克隆16S/trnI-trnA/23S片段,作为定点整合同源片段。然后将耐旱相关基因酵母海藻糖合酶基因tps1与水稻叶绿体16S rRNA基因的强启动子Prrn、烟草叶绿体基因psbA的终止子构建表达盒（Prrn-tps1-TpsbA-ter）,连同草丁膦抗性基因bar表达盒、卡那霉素抗性基因nptII和绿色荧光蛋白基因gfp构建的融和基因表达盒一起克隆到定点整合同源片段之间,构建高羊茅叶绿体的稳定表达载体gTKGB。通过基因枪轰击法将gTKGB转化到高羊茅幼嫩叶片中,用激光共聚焦扫描显微镜对GFP的表达情况进行检测分析。【结果】克隆的高羊茅叶绿体基因16S-trnI-trnA-23S在NCBI登录号为：DQ490947－DQ490950。构建的叶绿体表达载体gTKGB转化高羊茅幼嫩叶片,在叶绿体中有很好的GFP表达。【结论】高羊茅叶绿体表达载体gTKGB可用于高羊茅叶绿体转化。     

家蚕CDK11与RNPS1和9G8相互作用的鉴定

【目的】鉴定家蚕(Bombyx mori)CDK11与RNPS1和9G8的相互作用,为解析其是否参与前体RNA的剪接打下基础。【方法】利用家蚕基因组数据库Silk DB找到本研究克隆的家蚕基因序列,采用Primer 5.0进行引物设计,通过PCR技术克隆获得家蚕的两个重要剪接因子RNPS1和9G8,并构建具有不同抗原标签的过表达载体,采用NCBI检索并获得其他物种的相关序列,利用在线预测软件SMART进行结构域预测,采用Clustal X 1.83和GENEDOC 3.2预测同源序列,利用软件MEGA 6.0构建系统进化树,通过免疫荧光验证家蚕CDK11两种剪切体CDK11A和CDK11B分别与RNPS1和9G8的共定位情况,进一步通过免疫共沉淀验证CDK11A、CDK11B分别与RNPS1和9G8的相互作用。【结果】RNPS1的开放阅读框长为882 bp,编码293个氨基酸;9G8的开放阅读框长为531 bp,编码176个氨基酸;RNPS1和9G8都属于SR蛋白家族,具有该蛋白家族成员的一个富含丝氨酸/精氨酸(S/R)重复序列的RS结构域。同源序列比对表明,RNPS1和9G8都具有典型的RRM结构域,此外9G8还含有一个锌指结构域。系统进化分析显示,RNPS1聚类于无脊椎动物一支,与同为鳞翅目昆虫的斑点木蝶、黑脉金斑蝶等亲缘关系较近;9G8也聚类于无脊椎动物一支,与同为鳞翅目昆虫的黑脉金斑蝶、金凤蝶等关系较近。荧光共定位证明,RNPS1分别与CDK11A和CDK11B共定位于细胞核中,且具有点状共聚集现象;同时发现,RNPS1还具有胞质定位,点状聚集于核外周。而9G8分别与CDK11A、CDK11B在细胞核中也具有共定位现象。进一步通过免疫共沉淀发现,在所有的总蛋白和细胞裂解离心后的上清液样品中,均能检测到对应目的条带的表达,表明共转染的细胞均能正常表达目的蛋白,并且蛋白溶于上清。同时,在所有的共沉淀条带中,均能检测到对应目的条带,以上结果表明CDK11A、CDK11B均与RNPS1和9G8具有相互作用。【结论】家蚕RNPS1和9G8具有典型的RRM结构域,属于SR家族。CDK11A和CDK11B能够与RNPS1和9G8相互作用。     

草鱼MKLN1基因可变剪接和微卫星序列的多态性

MKLN1基因编码的muskelin蛋白主要通过其羧基端的kelch重复结构域与其它蛋白形成复合物行使功能。通过分析草鱼鳃组织的基因转录谱,发现其中的MKLN1基因存在(CT)15的微卫星序列,通过检测3个不同的草鱼种群,又发现(CT)9、(CT)10、(CT)11和(CT)13 4种多态性序列。通过克隆该段MKLN1基因组序列并与斑马鱼和红鳍东方鲀的MKLN1基因组序列比较,发现克隆得到的草鱼基因序列是由一个内含子和两个外显子组成,共编码144个氨基酸。通过进一步调查不同组织该基因的转录本,发现存在内含子剪接和内含子未被剪接的两个转录本。由于内含子序列未被剪接的转录本在内含子中存在TGA终止密码子,导致MKLN1基因翻译提前终止,翻译产物为失去部分kelch结构域的muskelin蛋白。相对于内含子正常剪接的转录本,没有被剪接内含子的转录本在各种组织中的表达量要明显低得多。简而言之,本研究发现了草鱼MKLN1转录本中的微卫星序列是来自未被剪切的内含子,而未被剪切的内含子导致该转录本编码部分kelch结构域缺失的muskelin蛋白。研究亮点:目前对于可直接锚定功能基因的I型微卫星标记研究很少。本文首次发现M...     

Research Progress of Alternative Splicing and Its Application in Livestock and Poultry Breeding

Alternative splicing(AS) refers to the process of selective splicing across exons or splice junctions during individual development or cell differentiation to produce tissue-or development-stage specific mRNA. Through AS process, organisms can produce many different protein isomers from a single gene and finally increase the proteome diversity. Therefore, AS plays an important role in the regulation of animal growth, development and physiological metabolism. This article not only summarized the types and the detection methods of alternative splicing, but also highlighted its impact on various economic traits of livestock and poultry, and prospected its application prospects in livestock and poultry breeding. This article could provide new ideas for the improvement of livestock and poultry breeding.     

Cytochrome b gene structure and consequences for resistance to Qo inhibitor fungicides in plant pathogens

The cytochrome b (cyt b) gene structure was characterized for different agronomically important plant pathogens, such as Puccinia recondita f sp tritici (Erikss) CO Johnston, P graminis f sp tritici Erikss and Hennings, P striiformis f sp tritici Erikss, P coronata f sp avenae P Syd & Syd, P hordei GH Otth, P recondita f sp secalis Roberge, P sorghi Schwein, P horiana Henn, Uromyces appendiculatus (Pers) Unger, Phakopsora pachyrhizi Syd & P Syd, Hemileia vastatrix Berk & Broome, Alternaria solani Sorauer, A alternata (Fr) Keissl and Plasmopara viticola (Berk & Curt) Berlese & de Toni. The sequenced fragment included the two hot spot regions in which mutations conferring resistance to QoI fungicides may occur. The cyt b gene structure of these pathogens was compared with that of other species from public databases, including the strobilurin-producing fungus Mycena galopoda (Pers) P Kumm, Saccharomyces cerevisiae Meyer ex Hansen, Venturia inaequalis (Cooke) Winter and Mycosphaerella fijiensis Morelet. In all rust species, as well as in A solani, resistance to QoI fungicides caused by the mutation G143A has never been reported. A type I intron was observed directly after the codon for glycine at position 143 in these species. This intron was absent in pathogens such as A alternata, Blumeria graminis (DC) Speer, Pyricularia grisea Sacc, Mycosphaerella graminicola (Fuckel) J Schr?t, M fijiensis, V inaequalis and P viticola, in which resistance to QoI fungicides has occurred and the glycine is replaced by alanine at position 143 in the resistant genotype. The present authors predict that a nucleotide substitution in codon 143 would prevent splicing of the intron, leading to a deficient cytochrome b, which is lethal. As a consequence, the evolution of resistance to QoI fungicides based on G143A is not likely to evolve in pathogens carrying an intron directly after this codon.     

可变剪接在肌肉发育中的作用研究进展

可变剪接发生在DNA转录为前体mRNA后，通过该过程可以使单个基因产生多个mRNA异构体和蛋白质亚型，增加了转录后加工过程中基因的信息多样性和蛋白质丰度。剪接体、剪接因子和其他RNA结合蛋白催化识别剪接位点和可变剪接外显子，参与调节可变剪接，从进化的角度来看，可变剪接在一定程度上为生物进化提供了驱动力。可变剪接作为组织特异性调节剂，参与肌肉发育的整个过程。在成肌细胞分化过程中，多聚嘧啶序列结合蛋白（polypyrimidine tract-binding protein，PTB）和RNA结合蛋白4（RNA-binding motif protein 4，RBM4）分别与原肌球蛋白的内含子多嘧啶序列和富含CU的内含子结合，共同调节α-原肌球蛋白的肌肉细胞特异性外显子选择的活性。RBM4可通过下调PTB表达并颉颃PTB在外显子选择中的活性来协同作用于肌细胞增殖分化的特异性剪接；肌球蛋白Ⅰ亚型通过可变剪接产生4种不同长度杠杆臂的蛋白质，影响肌肉的张力与拉伸激活；可变剪接可能是产生肌肉类型特异性的重要机制，选择性剪接产生具有不同催化动力学的肌球蛋白重链同工型，催化动力学差异地调节收缩性，从而影响肌纤维类型和肌肉功能。作者通过阐述可变剪接事件在肌肉发育及肌纤维形成过程中的主要作用，揭示可变剪接对肌肉发育的作用，以期为后续研究肌肉的发育调控机理提供参考依据。     

猪痘病毒TK、ORF121、ORF143基因缺失毒株的构建及其生物学特性的测定

为筛选猪痘病毒（SWPV）复制非必需区并测定其缺失株的生物学特性,本研究根据同源重组原理分别针对SWPV的TK（ORF063）、ORF121和ORF143基因设计引物。应用重叠延伸PCR技术拼接同源重组左右臂及EGFP筛选标记表达盒,将拼接片段分别转染到感染SWPV的PK15细胞。利用荧光显微镜观察标记含EGFP的单个蚀斑,筛选获取重组毒株。应用PCR及Western blotting对重组毒株进行鉴定。分别测定各毒株感染PK15细胞的蚀斑大小和皮下接种保育猪致病性。PCR结果表明,目的基因成功整合到相应位点,成功获得重组毒株rSWPV-TK、rSWPV-121和rSWPV-143;Western blotting结果显示,连续传代的重组毒株在PK15细胞上稳定表达外源蛋白。重组毒株在PK15细胞上形成的痘斑均小于亲本毒株,其中ORF121缺失株差异极显著（P<0.01）。各毒株均可导致保育猪痘斑形成,其中ORF121缺失株引起病变时间短,产生痘斑小,毒力较亲本下降。综上所述,本研究筛选到2个SWPV基因组中可供外源蛋白插入的复制非必需区ORF121和ORF143,为构建SWPV基因工程载体奠定了基础。     

Gene organization, alternate splicing and expression pattern of porcine visfatin gene

Visfatin is a newly discovered visceral fat-specific adipocytokine. It is upregulated in obesity and exerts insulin-mimetic effects in various tissues in human and mouse. We reported here the cloning and characterization of porcine visfatin, its three alternate splicing variants. Sequence analysis indicated that variant 1 is the predominant form among species, which contains an open reading frame of 1473 bp encoding a 52-kDa protein of 491 amino acids. While the other two variants were predicted to encode two 3' truncated proteins due to early termination. The nucleotide and amino acid sequences deduced from variant 1 were conservative across species. The porcine visfatin gene was composed of 11 exons at least and had exactly the same exon/intron structure as the human orthologs. Nested PCR showed that variants 1 and 3 were ubiquitously expressed in porcine tissues and that variant 2 was expressed in most tissues examined with exception of testis and liver. The discovery of the three variants of visfatin in porcine would be useful to the further investigation of the function of the visfatin gene.     

家兔骨骼肌肌球蛋白重链构成的电泳

[目的]探究家兔骨骼肌肌球蛋白重链构成的电泳的作用机制。[方法]应用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)检测成年家兔后肢肌胫前肌、腓肠肌和比目鱼肌的肌球蛋白重链异构体(MyHCs),采用大体解剖法依次取出家兔的每块骨骼肌,分别与样品缓冲液一起研磨成肌细胞匀浆,经高速冷冻离心后取出上清液,按照不连续SDS-聚丙烯酰胺小孔梯度凝胶电泳操作程序依次试验。[结论]该研究进一步证实3种快纤维型在成年啮齿动物肢体肌肉中的作用,但同源结构是否有这些肌纤维值得继续深入探索。