谷子肌动蛋白基因的克隆及序列分析

以谷子（Setaria italica）为材料,提取总RNA。根据植物肌动蛋白基因编码区的两端的保守序列设计了简并引物,用5'RACE方法扩增出了谷子肌动蛋白基因编码区序列。以豌豆肌动蛋白cDNA作探针进行的Southern杂交分析表明扩增出了目的基因。将所获得的片段克隆到T载体后进行测序,序列分析结果表明：谷子肌动蛋白基因的编码区长1131个核苷酸,编码了377个氨基酸;所得序列(命名为MIAc)与GenBank中注册的肌动蛋白基因序列的相似性均在60%以上,与其它肌动蛋白氨基酸序列的相似性达89%以上。根据高等植物肌动蛋白序列相似性重建了进化树,表明谷子肌动蛋白与水稻肌动蛋白异型体RAc2和RAc3之间的亲缘关系 最为密切,在进化过程中分化时间最为接近。     

白桦肌动蛋白(Actin)基因全长cDNA克隆与序列分析

以白桦(Betula platyphylla Suk.)次生木质部为材料,用改良CTAB方法提取总RNA。根据植物肌动蛋白(Actin)基因编码区的保守序列设计引物后进行RT-PCR,并采用RACE技术扩增出Actin基因全长序列。该基因cDNA全长1 785 bp,序列分析表明,该基因编码区1 134 bp,编码377个氨基酸,5′非编码区157 bp,3′非编码区495 bp。所得序列与GenBank中注册的其它植物肌动蛋白核苷酸序列的相似性均在80%以上,氨基酸序列的相似性高达96%以上。此基因已在GenBank注册（EU588981）。根据高等植物肌动蛋白相似性构建了进化树,表明白桦肌动蛋白与蓖麻肌动蛋白之间的亲缘关系最为密切,在进化中分化时间最为接近。     

兴安落叶松肌动蛋白基因的分离与序列分析

以兴安落叶松(Larix gmelinii)实生苗为材料,提取总RNA和基因组DNA;利用兼并PCR及RACE技术克隆肌动蛋白基因(Lg-act).序列分析的结果显示,该基因cDNA全长1662 bp,编码377个氨基酸;基因组DNA长度约为2564bp,包含4个内含子,5个外显子.所克隆的Lg-act cDNA序列与GenBank中注册的其它植物肌动蛋白核苷酸序列的相似性均在77％以上,氨基酸序列的相似性高达93％以上.根据高等植物肌动蛋白相似性构建的系统树显示,兴安落叶松肌动蛋白与挪威云杉肌动蛋白之间的亲缘关系较为密切,在进化中分化时间最为接近.     

斯达氏油脂酵母肌动蛋白基因克隆及序列分析

以产油真菌斯达氏油脂酵母(Lipomyces starkeyi AS 2.1560)为材料,提取总KNA,反转录成cDNA.根据真菌肌动蛋白(actin)保守核酸序列设计引物,PCR扩增后获得部分序列,再采用cDNA末端快速扩增技术获得了开放阅读框全长为1128 bP的cDNA序列,该序列编码蛋白质含375个氨基酸残基,等电点为5.49.同源性比较发现该基因(Accession No.EU258762)与其它已知真菌的actin基因相似性在75%以上,氨基酸序列相似性在90%以上.通过不同物种肌动蛋白进化树分析发现L.srarkeyi与亚罗解脂酵母(Yarrowia lipolytica)有较近的亲缘关系.该研究对探索L.starkeyi产油机制有一定的参考价值.     

茶树肌动蛋白基因（CsActin1）全长cDNA克隆与生物信息学分析

以茶树（Camellia sinensis）萌动芽为材料,根据茶树萌动芽芽抑制消减杂交文库中分离得到的肌动蛋白（actin）基因的5′-片段设计引物,利用3′-RACE技术克隆了其cDNA全长序列,该基因cDNA全长1 470 bp,命名为CsActin1（GenBank登录号HQ235647）。序列分析表明,CsActin1开放阅读框长1 134 bp,编码377个氨基酸,5′非编码区100 bp,3′非编码区236 bp。推测的蛋白质分子量为41.70 kD,等电点约为5.31,具有肌动蛋白家族的特征信号序列（YVGDEAQs.KRG和WIAKaEYDE）和肌动蛋白相关蛋白的特征信号序列（LLTEApLNPkaNR）。CsActin1与GenBank中注册的其它植物肌动蛋白核苷酸序列的相似性在80%以上,氨基酸序列相似性在95%以上。与其它植物肌动蛋白的进化树分析结果表明,茶树肌动蛋白与杨树的两个肌动蛋白间的亲缘关系最为密切。并对推导的蛋白结构进行了分析。     

球根白丝膜菌γ-actin基因的克隆及表达分析

根据真菌肌动蛋白(actin)基因保守区序列设计引物,用简并PCR法和RACE技术分离得到球根白丝膜菌(Leucocortinarius bulbiger)γ-肌动蛋白基因(Lb-act)的全长cDNA序列。该序列全长为1 357 bp,包含一个1 137 bp的开放阅读框(ORF),编码378个氨基酸,5'端非翻译区(5'UTR)92 bp,3'UTR长度128 bp。Port Param软件在线分析结果表明,该cDNA所编码的蛋白质理论等电点为5.12,相对分子质量为95.022 kD,具有真菌γ-actin基因3个保守特征序列。Blast同源性检索结果表明,Lb-act氨基酸序列与担子菌肌动蛋白序列有较高的相似性,其与双色蜡蘑的肌动蛋白氨基酸序列的亲缘关系最近。Lb-act基因在不同碳源及磷水平培养条件下表达量基本一致,验证了该基因作为分子内标的可靠性。     

白沙蒿肌动蛋白基因核心片段的克隆和序列分析

本研究以荒漠植物白沙蒿总RNA为模板,运用RT-PCR方法扩增出肌动蛋白基因核心序列.将获得的片段克隆到T载体后进行测序,序列分析表明:白沙蒿肌动蛋白基因核心片段长599 bp,编码198个氨基酸.将该序列在GenBank中注册,并与多种植物肌动蛋白序列进行同源性比较,发现该片段的核酸序列同源性在75%以上,氨基酸序列同源性在85%以上,具有高度保守性.     

中华大蟾蜍γ-肌动蛋白基因Bbgγ-Actin的克隆与生物信息学分析

旨在获得中华大蟾蜍肌动蛋白序列全长。以中华大蟾蜍(Bufo bufo gargarizans)耳后腺为材料,提取总RNA,通过转录组高通量测序快速得到中华大蟾蜍肌动蛋白基因(Bbg Actin)cDNA全长2 055 bp,通过RT-PCR技术对其ORF序列进行验证,并对Bbg Actin基因进行分析。序列分析表明,Bbg Actin基因开放阅读框长1 131 bp,编码376个氨基酸。通过BLAST P程序分析,所得序列与Gen Bank数据库中肌动蛋白基因序列相似度均在89%以上,氨基酸序列的相似性达90%以上。系统进化分析表明,Bbg Actin与γ-Actin聚为一类,所得中华大蟾蜍肌动蛋白属于γ-Actin。首次获得了中华大蟾蜍γ-Actin cDNA全长序列。     

多浆旱生植物霸王Actin基因片段的克隆及序列分析

根据其他植物Actin基因的保守序列设计一对简并性引物,以霸王叶片总RNA为模板,采用RT-PCR的方法扩增出Actin基因片段并克隆到PUCm-T载体.阳性克隆经PCR鉴定后进行测序,序列分析结果表明:该片段长598bp,编码198个氨基酸;所得序列与GenBank中注册的Actin基因序列的同源性均在82%以上,与其他肌动蛋白的氨基酸序列的同源性达91%以上.     

大熊猫生长激素受体(GHR) cDNA 的克隆与序列分析

根据已报道的若干物种GHR 基因cDNA 序列设计引物, 利用RT- PCR 技术首次从大熊猫肝脏组织总RNA中扩增出GHR 基因编码区全长cDNA 序列, 克隆于pGEM®-T 载体后进行测序和序列分析。结果表明,大熊猫GHR 的ORF为1 917 bp , 编码638 个氨基酸的前体蛋白, 由18 个氨基酸的信号肽和620 个氨基酸的成熟肽组成,与人、狗、猪GHR 结构相似, 大熊猫GHR 成熟肽由246 个氨基酸的胞外区、24 个氨基酸的跨膜区和350 个氨基酸的胞内区组成, 并具GHR 的特征性结构。序列相似性比较显示, 大熊猫GHR 与哺乳类GHR 具有69 %～93 %的高序列相似性, 与爬行类和鸟类的序列相似性也达到60 % , 而与鱼类的序列相似性较低, 仅为30 %左右。与其它哺乳动物GHR 相比, 大熊猫GHR 在氨基酸序列上也存在明显的特异性。     

Actin of Histriculus cavicola: characteristics of the highly divergent hypotrich ciliate actins.

A macronuclear gene-sized molecule carrying an actin gene from the hypotrich ciliate, Histriculus cavicola, was characterized. Southern blot analysis using a coding region probe suggested that actin in H. cavicola is encoded by a single gene. A comparison of the promoter regions indicated that the H. cavicola actin gene has a TATA box in the 5' flanking region in a position identical to those in other oxytrich ciliates. The coding sequence of this gene is not interrupted by any introns, and codes for a protein of 375 amino acid residues. This protein shares a high degree of similarity with other oxytrichid actins, and a relatively low similarity with actins from other eukaryotes. Comparative analyses of sequences indicated that most of the amino acid substitutions in hypotrich actins are found in surface loops, while the core structures are well-conserved. The sites that interact with DNase I and several regions involved in actin-actin contact have diverged considerably in hypotrich actins, while nucleotide-binding sites are the best-conserved interaction motif.     

Actin Gene Sequence from Euglena gracilis

ABSTRACT The full length coding sequence of the Euglena gracilis actin gene was determined by RT-PCR of Euglena gracilis mRNA. Conserved regions in the actin amino acid sequence were used as guides for the synthesis of degenerate primers. Sequence was obtained for 1.238 nucleotides, of which 1.131 were coding for 377 amino acids. Sequence comparisons showed a similarity with other actins of 56% to 80%. Even though most of the actin amino acid sequence was conserved, some regions showed high divergence, i.e. the DNase I-binding loop at the N-terminal region. The construction of a phylogenetic tree based on actin sequences from different organisms placed Euglena gracilis in a cluster with Trypanosoma brucei and Leishmania major.     

石蒜Mg^2＋转运体基因LrMGT的克隆与分析

从石蒜〔Lycoris radiata（L’Hér.）Herb.〕叶片全长cDNA文库中克隆获得Mg^2＋转运体（MGT）基因LrMGT。序列分析结果显示：LrMGT基因的cDNA序列全长1 726 bp,其中开放阅读框（ORF）长度921 bp,编码306个氨基酸。石蒜LrMGT基因编码的氨基酸序列的理论相对分子质量为33 635,理论等电点为pI 5.14,为疏水性膜蛋白,不具有信号肽。序列比对结果表明：石蒜LrMGT基因编码的氨基酸序列与小米〔Setaria italica（Linn.）Beauv.〕、水稻（Oryza sativa Linn.）和拟南芥〔Arabidopsis thaliana（Linn.）Heynh.〕等植物的MGT基因编码的氨基酸序列的相似性较高,相似度达到72%~76%;石蒜LrMGT基因与其他植物MGT基因编码的氨基酸序列的保守区域较大,均具有较高的保守性。在NJ系统树上石蒜LrMGT基因编码的氨基酸序列与禾本科（Gramineae）植物二穗短柄草〔Brachypodium distachyum（Linn.）Beauv.〕、水稻、高粱〔Sorghum bicolor（Linn.）Moench〕和小米MGT基因编码的氨基酸序列聚为同一个分支,表明它们可能具有较近的进化关系。实时荧光定量PCR结果表明：石蒜LrMGT基因在根和鳞茎中的相对表达量较高,在叶片和花中的相对表达量较低,具有明显的组织特异性。     

Amino acid sequence of Acanthamoeba actin

By amino acid sequence studies, only one form of cytoplasmic actin was detected in Acanthamoeba castellanii. Its amino acid sequence is very similar to the sequences of Dictyostelium and Physarum actins, from which Acanthamoeba actin differs in only nine and seven residues, respectively, including the deletion of the first residue. Acanthamoeba actin is unique in containing a blocked NH2-terminal neutral amino acid (glycine), while all other actins sequenced thus far have a blocked acidic amino acid (aspartic or glutamic) at the NH2 terminus. Acanthamoeba actin is also unique in that it contains an N epsilon-trimethyllysine residue at position 326. Like other actins, Acanthamoeba actin contains an NT-methylhistidine residue at position 73. The protein sequence is in complete agreement with the sequence derived from the nucleotide sequence of an expressed actin gene.     

Expression of the genes coding for the skeletal muscle and cardiac actions in the heart

Several types of evidence indicate that the gene coding for the skeletal muscle actin is expressed in the rat heart: 1) A recombinant plasmid containing an insert with a nucleotide sequence identical to that of the homologous region of skeletal muscle actin gene was isolated from a cDNA library prepared on rat cardiac mRNA template. 2) Using specific probes it was found that the hearts of newborn rats contain a significant amount of skeletal muscle actin mRNA. The quantity of this mRNA in the heart decreases during development. 3) The skeletal muscle actin gene is DNAase I sensitive in nuclei from rat heart tissue. A plasmid containing a cDNA insert homologous to a part of the cardiac actin mRNA was isolated and sequenced. It was found that in spite of the great similarity between the amino acid sequence of the skeletal muscle and cardiac actins, the nucleotide sequences of the two mRNAs are considerably divergent. There is only limited sequence homology between the 3' untranslated regions of the two mRNAs. However, there is an extensive sequence homology between the 3' untranslated regions of the rat and human cardiac mRNAs, suggesting a functional role for this region of the gene or mRNA.     

A Ser–Gly substitution in plastid-encoded photosystem II D1 protein is responsible for atrazine resistance in foxtail millet (<Emphasis Type="Italic">Setaria italica</Emphasis>)

A foxtail millet (Setaria italica L. Beauv.) line resistant to atrazine was obtained through interspecific hybridization between wild S. viridis L. Beauv. and cultivated S. italica. The resistance was proved to be controlled by a chloroplast-inherited gene and it has further been utilized in foxtail millet production. However, the sequence information of the putative atrazine resistance gene, psbA in foxtail millet’s chloroplast genome encoding photosystem II D1 protein (32 kDa thylakoid membrane protein) (photosystem Q_B protein) and the mutation site responsible for the resistance are not known. In this paper the psbA sequences of six atrazine susceptible/resistant foxtail millet varieties were obtained and compared. The results indicated that there was only one amino acid difference between susceptible and resistance gene, resulting from a single base substitution. It was concluded that a mutant allele of photosystem II protein D1 encoding a Gly residue instead of a Ser residue at position 264 is a major gene of resistance to atrazine. Moreover, the phylogenetic tree based on the psbA coding region of thirty-five plant species was carried out. The phylogenetic relationship between S. italica and other plants and the related evolutionary issues were discussed and it was suggested that psbA sequences could be used in phylogenetic studies in plants. Xiaoping Jia and Jincheng Yuan have equal contribution.     

The Molecular Evolution of Actin

We have investigated the molecular evolution of plant and nonplant actin genes comparing nucleotide and amino acid sequences of 20 actin genes. Nucleotide changes resulting in amino acid substitutions (replacement substitutions) ranged from 3-7% for all pairwise comparisons of animal actin genes with the following exceptions. Comparisons between higher animal muscle actin gene sequences and comparisons between higher animal cytoplasmic actin gene sequences indicated less than 3% divergence. Comparisons between plant and nonplant actin genes revealed, with two exceptions, 11-15% replacement substitution. In the analysis of plant actins, replacement substitution between soybean actin genes SAc1, SAc3, SAc4 and maize actin gene MAc1 ranged from 8-10%, whereas these members within the soybean actin gene family ranged from 6-9% replacement substitution. The rate of sequence divergence of plant actin sequences appears to be similar to that observed for animal actins. Furthermore, these and other data suggest that the plant actin gene family is ancient and that the families of soybean and maize actin genes have diverged from a single common ancestral plant actin gene that originated long before the divergence of monocots and dicots. The soybean actin multigene family encodes at least three classes of actin. These classes each contain a pair of actin genes that have been designated kappa (SAc1, SAc6), lambda (SAc2, SAc4) and mu (SAc3, SAc7). The three classes of soybean actin are more divergent in nucleotide sequence from one another than higher animal cytoplasmic actin is divergent from muscle actin. The location and distribution of amino acid changes were compared between actin proteins from all sources. A comparison of the hydropathy of all actin sequences, except from Oxytricha, indicated a strong similarity in hydropathic character between all plant and nonplant actins despite the greater number of replacement substitutions in plant actins. These protein sequence comparisons are discussed with respect to the demonstrated and implicated roles of actin in plants and animals, as well as the tissue-specific expression of actin.     

豌豆卷须肌动蛋白Ⅱ类异型体cDNA克隆的序列分析

分析１５个豌豆卷须肌动蛋白ｃＤＮＡ 克隆的限制性内切酶图谱,发现在豌豆卷须中至少存在三类肌动蛋白异型体,分别命名为Ⅰ类（ＰＥＡｃ Ⅰ）、Ⅱ类（ＰＥＡｃ Ⅱ）和Ⅲ类（ＰＥＡｃ Ⅲ）异型体．三类异型体的克隆数目分别为１０、４和１个,表明三类异型体在豌豆卷须中的表达是不同的,很可能具有组织或发育阶段的特异性．对Ⅱ类异型体的三个ｃＤＮＡ 克隆ＰＥＡｃ３、ＰＥＡｃ９和ＰＥＡｃ１１进行了全序列测定,所测定的序列已被ＧｅｎＢａｎｋ 数据库所接受．测序结果表明,ＰＥＡｃ３、ＰＥＡｃ９和ＰＥＡｃ１１的序列长度分别为１５５０、１６８０和１０９１个核苷酸,其中编码区长１１３４个核苷酸,编码的氨基酸长度为３７７（ＰＥＡｃ１１缺少编码氨基端前９６个氨基酸的核苷酸序列）．三个克隆的核苷酸序列完全相同,差别仅在于３′非翻译区的长度不同,即ｐｏｌｙ（Ａ）的加入位点不同．这说明它们可能是由同一基因转录而来,但转录后的加工过程不同．豌豆卷须中肌动蛋白基因ｐｏｌｙ（Ａ）加入位点的使用,可能与组织或发育的特异性表达有关．此外,豌豆卷须肌动蛋白三类异型体之间的核苷酸序列同源性为８０％ ,氨基酸序列同源性为９４％ ．