关于青岛文昌鱼SoxB2和SoxC基因的研究：进化保守性分析

大多数Sox基因在细胞命运决定和分化过程中起着重要的作用.本研究分离了青岛文昌鱼（Branchiostoma belcheri）的SoxB2和SoxC基因,对其预测的蛋白序列进行了序列比对、进化树分析以及基因时空表达分析.结果显示,文昌鱼SoxB2和SoxC虽然在进化树中不属于脊椎动物SoxB2和SoxC进化支,但它们在进化过程中基因的结构和表达部位保守性很高,都具有保守的HMG结构域;这两个基因在胚胎发育早期都在神经外胚层和原肠腔壁表达,而在成体中不仅存在于神经索中,还存在于肠、肝盲囊、鳃和卵母细胞中.SoxB2和SoxC基因表达部位的相似说明两者可能在中枢神经系统、性腺及免疫系统的胚胎发育和成体的免疫应答过程中共同起作用;两者与脊椎动物同源基因的表达部位相同说明这两个基因在进化过程中可能功能保守.     

白氏文昌鱼FADD的克隆及功能研究

Fas死亡结构域相关蛋白(Fas-associated death domain protein,FADD)是死亡信号转导通路中的连接蛋白,在脊椎动物中其结构和功能都很保守.本文首次克隆了头索动物白氏文昌鱼(Branchiostoma belched)FADD(bbFADD)的cDNA和基因组DNA序列.bbFADD cDNA全长1239 bp,编码217个氨基酸.与脊椎动物的FADD一样,bbFADD含有N端的死亡效应结构域(Death Effector Domain,DED)和C端的死亡结构域(Death Domain,DD).bbFADD氨基酸序列的第33位氨基酸苯丙氨酸在进化过程中相对保守,此苯丙氨酸在FADD自我相互作用中具有重要作用.哺乳类的FADD基因编码区含有两个外显子,而bbFADD基因含有3个外显子.一般认为头索动物处在无脊椎动物进化到脊椎动物的中间过渡阶段,但基于FADD氨基酸序列的系统进化树和同源性分析显示,文昌鱼与海胆的亲缘关系更近.bbFADD在HeLa细胞中超表达能够引起HeLa细胞的凋亡,暗示bbFADD可能能够在人类细胞凋亡通路中起作用,推测凋亡系统在生物进化过程中相当保守.     

文昌鱼RACK1基因在胚胎发育中的表达

脊椎动物活化的(蛋白)激酶C受体1 (receptor for activated C-kinase 1, RACK1)在胚胎发育过程中起着至关重要的作用. 克隆了RACK1基因在青岛文昌鱼(Branchiostoma belcheri)中的同源基因AmphiRACK1, 对其进行了系统的进化学分析, 并研究了该基因在正常胚胎发育过程中和经LiCl处理胚胎发育中的时空表达图式. 系统进化学分析结果表明, 文昌鱼RACK1位于脊椎动物进化枝的基部. 在正常胚胎发育中, AmphiRACK1基因在脑泡、神经管和体节中都有明显的表达. 在经LiCl处理的胚胎中, 该基因在体节中的分节型表达变模糊, 表达下调; 在脑泡和神经管中的表达下调甚至消失. 此外, 在成体文昌鱼的轮器、鳃血管、肝盲囊和肠上皮以及精巢中都检测到AmphiRACK1基因不同程度的表达.     

文昌鱼一个GATA基因在胚胎发育中的表达图式(英文）

GATA基因在脊椎动物和非脊椎动物的发育中行使重要的功能,该家族的成员在进化上也是非常保守的。脊椎动物的GATA基因分为两个亚群：GATA1/2/3和GATA4/5/6。通过生物信息分析,在文昌鱼的基因组中找到了3个GATA基因：一个GATA1/2/3亚家族基因,两个GATA4/5/6亚家族基因;还找到一个类GATA基因。还克隆了白氏文昌鱼(Branchiostoma belcheri)GATA123的一段序列,并研究了它在早期胚胎发育中的表达图式。结果表明GATA123在原肠胚的中内胚层表达,而在神经胚晚期和幼体早期,GATA123在脑泡和消化道中部区域表达。这种表达模式与头部发育的重要基因Otx相类似。结果提示在文昌鱼脑泡的发育过程中GATA123和Otx很可能共同发挥着重要的作用。     

ycaCR基因在文昌鱼早期胚胎表达研究

文昌鱼作为现存的与脊椎动物最接近的无脊椎动物,一直被作为研究生物进化和胚胎发育的典型材料.利用整体原位杂交方法对从文昌鱼肠cDNA文库克隆到的ycaCR基因进行基因的胚胎表达模式研究,结果显示该基因在早期胚胎发育阶段没有表达,在2天幼虫的原始消化道表达,暗示ycaCR基因可能在原始消化道内发挥生物学作用.     

文昌鱼AmphiDC-like的全长cDNA克隆及其表达模式分析

本文旨在克隆文昌鱼树突样蛋白基因AmphiDC-like,采用实时 PCR法和原位分子杂交法对其表达模式进行分析.从文昌鱼神经胚cDNA文库测序得到的ESTs中筛选得到该基因片段,通过引物步移直接测序的方法,克隆得到其cDNA全长序列,对其推测的氨基酸序列进行同源性分析发现,该基因产物具有树突样细胞蛋白共有的保守区,并与多种生物的树突状细胞蛋白具有高度同源性,其中与脊椎动物的树突样细胞蛋白同源性较高. 实时 PCR结果显示, AmphiDC-like在文昌鱼受副溶血性弧菌(Vibrio parahemolyticus,Vp)感染后6 h到48 h表达均上调,并通过原位分子杂交技术观察到,该基因在文昌鱼鳃和消化道中有表达,为深入研究其功能奠定了基础.     

基于COXI基因的太平洋西岸文昌鱼地理种群分析

文昌鱼是进化发育研究的重要模式动物,目前实验材料均采自野外.因此,对其进行正确的物种鉴定和地理种群的遗传分化分析十分必要.该研究扩增并测定了COX 1基因部分序列,结合NCBI数据库中的COX 1序列信息,对太平洋西岸文昌鱼的种类和地理种群分化情况进行了分析.结果表明,马来文昌鱼(Branchiostoma malayanum)、白氏文昌鱼(B.belcheri)和日本文昌鱼(B.japonicum)这3个种之间的遗传差异很大,再次证实3个物种的有效性,同时提出应当审慎对待NCBI数据库中文昌鱼的种名标注;太平洋西岸文昌鱼属Branchiostoma的3种文昌鱼群体遗传多样性均处于较高水平,同一物种的不同地理种群间没有出现明显的遗传分化,反映了海洋动物的基因交流较容易,不同海域隔离较弱.     

trp14基因在文昌鱼早期发育阶段的时空表达及免疫活性初步研究

探讨文昌鱼trp14 (thioredoxin-related protein of 14 kD)基因在文昌鱼早期发育阶段的时空表达模式及其免疫活性.利用整体原位杂交技术研究trp14基因在文昌鱼早期发育阶段的时空表达模式;通过半定量RT-PCR方法分析trp14基因在低温胁迫和免疫药物刺激下的mRNA表达变化.trp14基因在文昌鱼2 d幼虫的原始消化道表达,呈现时空特异的表达模式;低温可以增强trp14基因的表达,而免疫刺激药物LPS和LTA则降低trp14基因的表达量.文昌鱼trp14基因在胁迫条件下表达量发生变化,暗示其可能参与氧化压力变化引起的免疫反应.     

文昌鱼AmphiMef2基因的特征及其发育表达

肌细胞增强因子2(MEF2)属转录因子MADS家族成员, 它能控制脊椎动物肌肉特异基因的表达, 但在无脊椎动物中, 并非所有的Mef2基因都是肌肉发育所必需的. 在青岛文昌鱼(Branchiostoma belcheri)中首次克隆到一个全长的cDNA, 定名为AmphiMef2. 其编码的氨基酸序列具有高度保守的MADS和MEF2结构域, 与脊椎动物同源蛋白相应区域的氨基酸一致性高达95.3%. 原位杂交结果表明, AmphiMef2首先在早神经胚的预定体节中胚层中表达, 之后在体节和未分节的预定体节中胚层中表达. 36 h幼虫期, 只在后部体节中检测到它的表达. 48 h幼虫 期, AmphiMef2的表达区域转移到口前窝(一个与脊椎动物腺垂体同源的器官), 且持续表达到至少 72 h幼虫期. 实验结果提示, 文昌鱼AmphiMef2可能不但参与肌肉发生, 而且可能在口前窝的发育或功能发挥中起作用.     

文昌鱼分类学研究及展望

文昌鱼是最接近脊椎动物直接祖先的现生动物,在脊椎动物起源与演化研究中占有极其重要的位置。近年来,对文昌鱼的研究已引起越来越多的科学家的兴趣,然而作为生命科学研究的重要基础,这类动物的分类学研究相对滞后。依据已有的中国文昌鱼资源调查资料,中国沿海文昌鱼的分布应当十分广泛,即只要有适合文昌鱼栖息的沙滩,均有文昌鱼分布的可能。根据目前的分类学研究成果和动物命名法中的优先权原则,建议将产于青岛等地的文昌鱼种名Brnachiostoma belcheri tsingtauense订正为B.japonicus,南方的文昌鱼保留其原种名B.belcheri。由此,目前分布在中国沿海的鳃口文昌鱼属(Branchiostoma)至少有2种,侧殖文昌鱼属(Epigonichthys)有1～3种,漂浮文昌鱼(Amphioxides pelagicus)1种。DNA分子标记技术在文昌鱼分类学研究中将会发挥更大的作用。     

Characterization and expression of p23 gene in the amphioxus Branchiostoma belcheri

The cDNA AmphiP23, encoding an amphioxus p23, was identified from the gut cDNA library of amphioxus Branchiostoma belcheri. It contains a 513 bp open reading frame corresponding to a deduced protein of 170 amino acids. Phylogenetic analysis shows that vertebrate and invertebrate p23/p23-like proteins are each grouped together, with AmphiP23 falling at the base of vertebrate p23/p23-like clade, suggesting that the divergence of vertebrate and invertebrate p23 genes probably occurs prior to the split of invertebrate/vertebrate from a common ancestor around 550 million years ago. Northern blotting reveals a ubiquitous expression pattern of AmphiP23 in all adult tissues examined, while whole mount in situ hybridization demonstrates a tissue- and stage-specific expression pattern of AmphiP23 in developing embryos and larvae. Presumably, the ubiquitous expression pattern of AmphiP23 in adult amphioxus represents the ancestral type of p23 gene prior to its split to human paralogs p23 and tsp23, while the tissue- and stage-specific expression pattern during early embryonic development implicates a role of AmphiP23 in anterior/posterior patterning.     

Identification and tissue-specific expression of amphioxus GM2 activator protein gene from amphioxus Branchiostoma belcheri.

An amphioxus cDNA, AmphiGM2AP, encoding GM2 activator protein was isolated from the gut cDNA library of Branchiostoma belcheri. It is 907 bp long, and its longest open reading frame codes for a precursor protein consisting of 242 amino acid residues with a signal peptide of 14 amino acids. The deduced amino acid sequence includes a conserved domain typical of GM2APs between residues 53 and 224, a single N-linked glycosylation site at position 65 and 8 conserved cysteines. Phylogenetic analysis showed that amphiGM2AP forms a club together with invertebrate GM2APs, indicating that AmphiGM2AP is evolutionarily closely related to invertebrate GM2APs rather than vertebrate ones. Both Northern blotting and in situ hybridization histochemistry analyses revealed a tissue-specific expression pattern of AmphiGM2AP in adult amphioxus with the strongest expression in the digestive system, which is in contrast to the widespread expression pattern of human, mouse and sheep GM2AP genes. It is suggested that AmphiGM2AP is possibly involved in the take-in of digested food components like lipid molecules.     

文昌鱼sfy1基因的克隆及其在早期发育中的表达

文昌鱼是公认现存最接近于脊椎动物的一种头索动物,具有与脊椎动物相似但简单得多的身体图式[1],因而是研究脊椎动物发育机制起源和进化的宝贵材料,也是发育生物学的经典实验模型之一.近年来,人们在对果蝇和脊椎动物发育分子机制的研究取得了一系列重大突破之后,利用发育调控基     

Cloning, expression, purification, and characterization of AmphiTip30, a member of short-chain dehydrogenases/reductases family from the amphioxus Branchiostoma belcheri tsingtauense

In this paper we describe the cloning, expression and identification study of the TIP30 gene from amphioxus (Branchiostoma belcheri). The amphioxus TIP30 cDNA is comprised of 1499 bp and is translated in one open-reading frame to give a protein of 237 amino acids, with a predicted 23 amino acids signal peptide, a 147 bp 5'-UTR and a 638 bp 3'-UTR. A multiple alignment of TIP30 from amphioxus with other known TIP30 sequences shows the conservation of most amino acid residues involved in the peculiar structural domains found within TIP30's. Phylogenetic analysis places AmphiTIP30 at the base of the phylogenetic tree, suggesting that AmphiTIP30 is the archetype of the vertebrate TIP30 genes. We express the amphioxus TIP30 gene in Escherichia coli. driven by T7 promoter. The recombinant amphioxus TIP30 protein was purified by HisTrap affinity column. Subsequently, the binding constant and enzyme activity was mensurated. Western blot and immunohistochemistry analysis confirmed that amphioxus has a native molecular mass of approximately 26 kDa, and TIP30 was strongly expressed in ovary. Finally, the initial function of TIP30 is discussed.     

Characterization, evolution and tissue-specific expression of AmphiCalbin, a novel gene encoding EF-hand calcium-binding protein in amphioxus Branchiostoma belcheri

An amphioxus full-length cDNA, AmphiCalbin, encoding a novel EF-hand calcium-binding protein （EFCaBP）, was isolated from the gut cDNA library of amphioxus Branchiostoma belcheri. It consists of 1321 bp with a 636 bp open reading frame encoding a protein of 211 amino acids with a molecular mass of approximately 24.5 kDa. The phylogenetic analysis offers two interesting inferences. First, AmphiCalbin clusters with a group of unnamed EFCaBPs that are differentiated from other identified EFCaBPs. Second, AmphiCalbin falls at the base of the vertebrate unnamed EFCaBPs clade, probably representing their prototype. This is also corroborated by the fact that AmphiCalbin has an exon-intron organization identical to that of vertebrate unnamed EFCaBP genes. Both tissue-section in situ hybridization and whole-mount in situ hybridization prove a tissue-specific expression pattern of AmphiCalbin, with high levels of expression in the digestive system and gonads. It is proposed that AmphiCalbin might play a role in the digestive system and gonads. These observations lay the foundation for further understanding of the function of the unnamed EFCaBPs.     

Ribosomal Protein Genes S23 and L35 from Amphioxus Branchiostoma belcheri tsingtauense： Identification and Copy Number

The complete cDNA and deduced amino acid sequences of the ribosomal proteins S23 （AmphiS23） and L35 （AmphiL35） from amphioxus Branchiostoma belcheri tsingtauense were identified in this study. AmphiS23 cDNA is 546 bp long and encodes a protein of 143 amino acids. It has a predicted molecular mass of 15,851 Da and a pI of 10.7. AmphiL35 cDNA comprises 473 bp, and codes for a protein of 123 amino acids with a predicted molecular mass of 14,543 Da and a pI of 10.8. AmphiS23 shares more than 83% identity with its homologues in the vertebrates and more than 84% identity with those in the invertebrates. AmphiL35 is more than 63% identical to its counterparts in the vertebrates and more than 52% identical to those in the invertebrates. Southern blot analysis demonstrated the existence of 1-2 copies of the S23 gene and 2-3 copies of the L35 gene in the genome of amphioxus B. belcheri tsingtauense. This is in sharp contrast to the presence of 6-13 copies of the S23 gene and 15-17 copies of the L35 gene in therat genome. It is clear that the housekeeping genes like S23 and L35 underwent a large-scale duplication in the vertebrate lineage, reinforcing the gene/genome duplication hypothesis.     

Tissue- and stage-specific expression of a fatty acid binding protein-like gene from amphioxus Branchiostoma belcheri

A cDNA clone encoding an amphioxus fatty acid binding protein-like (AmphiFABPL) protein was isolated from a gut cDNA library of Branchiostoma belcheri. It contained a 423 bp open reading frame corresponding to a deduced protein of 140 amino acids with a predicted molecular mass of approximately 15.9 kDa. Phylogenetic analysis showed that AmphiFABPL fell outside the vertebrate clade of fatty acid binding proteins (FABPs), being positioned at the base of the chordate lineage, and was almost equally homologous to various vertebrate FABPs, suggesting that it may be the archetype of vertebrate FABPs. Both northern blotting and in situ hybridization analyses demonstrated that AmphiFABPL was expressed in the hepatic caecum and hind-gut, and although at a much lower level, it was also present in the endostyle, ovary and testis. In addition, whole-mount in situ hybridization revealed that AmphiFABPL was initially expressed in the posterior two thirds of the primitive gut, including the mid-gut where the hepatic caecum will form later, in 2-day larvae. The expression pattern is closely similar to that of the L-FABP and I-FABP genes in vertebrates, supporting the hypothesis that the hepatic caecum in the amphioxus is homologous to the vertebrate liver.