通过Cyt b基因同源序列比较评估厦门文昌鱼的分类学地位

白氏文昌鱼Branchiostomabelcheri (Gray)在我国和日本沿海均有分布 ,由于南、北方文昌鱼形态学上有一定差异 ,且二者间存在一些过渡类型 ,其分类地位问题仍有待进一步澄清。本文测定了厦门欧厝海域产的文昌鱼mtDNACytb基因序列 ,并与日本产的文昌鱼以及另外产于大西洋的两种文昌鱼Cytb基因序列比较。分子系统学分析结果表明 :厦门欧厝海域产的文昌鱼与日本产的文昌鱼平均遗传距离为 2 1 12 % ,达到了种间分化的水平 ;经过对已有文献和文昌鱼地理分布的综合分析 ,作者建议将原来的白氏文昌鱼青岛亚种B belcheritsingtauense提升为种 ,南、北方所产文昌鱼分别作为两个独立的种存在 ,即南方的B belcheri (Gray)和北方的B tsingtauenseTchangetKoo     

Functional conservation and innovation of amphioxus RIP1-mediated signaling in cell fate determination

Recently, receptor interacting protein (RIP)-1 has been recognized as an intracellular sensor at the crossroads of apoptosis, necroptosis, and cell survival. To reveal when this crucial molecule originated and how its function in integrating stress signals evolved, in this study we report on two RIP1 homologs in Chinese amphioxus (Branchiostoma belcheri tsingtauense), designated B. belcheri tsingtauense RIP1a and B. belcheri tsingtauense RIP1b. Phylogenetic analysis indicates that they are generated by domain recombination and lineage-specific duplication. Similar to human RIP1, both B. belcheri tsingtauense RIP1a and B. belcheri tsingtauense RIP1b activate NF-κB in a kinase activity-independent manner and induce apoptosis through the Fas-associated death domain protein-caspase cascade. Moreover, we found that the natural point mutation of Q to I in the RIP homotypic interaction motif of B. belcheri tsingtauense RIP1a provides negative feedback for amphioxus RIP1-mediated signaling. Thus, our study not only suggests that RIP1 has emerged as a molecular switch in triggering cell death or survival in a basal chordate, but also adds new insights into the regulation mechanisms of RIP1-related signaling, providing a novel perspective on human diseases mediated by RIP1.     

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。     

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.     

厦门两种文昌鱼染色体的制备与观察

文吕鱼的进化地位十分重要,对其染色体的研究在进化和比较基因组学方面有重要意义.然而文昌鱼的染色体制备困难,使研究受到了限制.本文介绍了一种改良的义昌鱼胚胎细胞染色体标本制备方法,以及用文昌鱼成体再生细胞制备染色体,首次获得了文昌鱼体细胞中期染色体标本,并观察了厦门2种文昌鱼的染色体,其中白氏文吕鱼(Branchiostoma belcheri)二倍体2n=40,日本文昌鱼(B.japonicum)二倍体2n=36.再次从细胞分类学角度证实白氏文昌鱼和日本文昌鱼作为两个独立物种的分类地位.     

Ribosomal proteins L34 and S29 of amphioxus Branchiostoma belcheri tsingtauense: cDNAs cloning and gene copy number

The complete cDNA and deduced amino-acid sequences of ribosomal proteins L34 (AmphiL34) and S29 (AmphiS29) from the amphioxus Branchiostoma belcheri tsingtauense were identified in this study. The AmphiL34 cDNA is 435 nucleotides in length and encodes a 118 amino-acid protein with calculated molecular mass of 13.6 kDa. It shares 53.6-67.5% amino-acid sequence identity with its eukaryotic counterparts including human, mouse, rat, pig, frog, catfish, fruit fly, mosquito, armyworm, nematode and yeast. The AmphiS29 cDNA comprises 453 nucleotides and codes for a 56 amino-acid protein with a calculated molecular mass of 6.6 kDa. It shows 66.1-78.6% amino-acid sequence identity to eukaryotic S29 proteins from human, mouse, rat, pig, zebrafish, seahorse, fruit fly, nematode, sea hare and yeast. AmphiL34 contains a putative nucleolar localization signal, while AmphiS29 has a zinc finger-like domain. A phylogenetic tree deduced from the conserved sequences of AmphiL34 and AmphiS29 and other known counterparts indicates that the positions of AmphiL34/AmphiS29 are intermediate between the vertebrate and invertebrate L34/S29. Southern blot analysis demonstrates the presence of one copy of the L34 gene and 2-3 copies of the S29 gene in the genome of the amphioxus B. belcheri tsingtauense. This is in sharp contrast to the existence of 7-9 copies of the L34 gene and 14-17 copies of the S29 gene in the rat genome. These date suggest that housekeeping genes like AmphiL34 and AmphiS29 have undergone large-scale duplication in the chordate lineage.     

EST analysis of mRNAs expressed in neurula of Chinese amphioxus

Amphioxus, a cephalochordate, is the closest living relative to the vertebrates. In order to investigate the molecular mechanisms of the early embryogenesis of amphioxus, we constructed a neurula embryo cDNA library of Chinese amphioxus (Branchiostoma belcheri tsingtauense) and generated 5235 expressed sequenced tags in the present study. The initial ESTs consisted of 638 clusters and 1855 singletons, which revealed approximately 2493 unique genes in the data set. Of these sequences, 35.52% ESTs matched to known genes, 12.76% matched to other ESTs, and 51.71% had no match to any known sequences in GenBank. Interestingly we found homologous genes related to neural development and human disease. Bioinformatic analysis showed the direct evidence that the gene homologue found only in vertebrates in previous studies also exists in the amphioxus genome. This study provides a preliminary view of the gene information involved in the development of neurula embryos of Chinese amphioxus and helps our understanding of vertebrate evolution at gene level.     

Branchiostoma japonicum and B. belcheri are distinct lancelets (Cephalochordata) in Xiamen waters in China

Lancelets in Xiamen were reported as Branchiostoma belcheri in 1932, and subsequently were believed to comprise a single species. However, recent studies revealed that Xiamen lancelets actually represent two species, B. belcheri and B. japonicum. We observed thousands of lancelets from Xiamen beach to recognize these two species. Our observations showed that at least three morphological characters distinguish them: 1) the rostral fin is slightly round with the end obtuse in B. belcheri but elliptic with the end cuspate in B. japonicum; 2) the number of preanal fin-chambers is more than 80 in B. belcheri but less than 64 in B. japonicum, and the chambers are slender in the former but stout in the latter; 3) the caudal fin of B. belcheri is narrower than that of B. japonicum, and the angle between the dorsal and super-caudal fins, and between preanal and sub-caudal fins, is obtuse in B. belcheri but acute in B. japonicum. We also provide some ecological and distributional evidence to support the conclusion that there are two separate species in Xiamen waters.     

文昌鱼的实验室繁育及子二代获得

文昌鱼特殊的进化地位、简单的器官系统和终生透明的躯体等特征,使其很有希望成为一个新型实验室模式动物。要实现文昌鱼的模式动物化,实验室内全人工条件下繁殖是关键的第一步。为此,我们于2003年9月和2004年4月两次采集产于厦门海域的2种文昌鱼,开展实验室内养殖研究。经过3年多的持续实验室养殖,继2005年夏季于实验室内繁殖出子一代文昌鱼后,又在2006年夏季成功获得了这两种文昌鱼的子二代,初步实现文昌鱼在实验室内的全人工养殖。对子代文昌鱼养殖的初步观察发现,不同水温对生长发育速度有一定影响,提示有可能通过水温控制实现文昌鱼一年多次产卵的目的。目前这两种文昌鱼子二代幼体已完成变态,进入亚成体生长发育阶段,其体长分别已达14.6mm(日本文昌鱼Branchiostomajaponicum)和6.5mm(白氏文昌鱼B.belcheri)。     

Continuous culture of two lancelets and production of the second filial generations in the laboratory

The lancelet is considered to be a promising laboratory model animal. To establish laboratory colonies of lancelet, we collected parental lancelets of Branchiostoma belcheri and B. japonicum (previously named as B. belcheri tsingtauense) with fully developed gonads from Xiamen Rare Marine Creature Conservation Areas (Fujian, China) on dates just before their spawning in the field in 2005. Those parental lancelets spawned spontaneously in the laboratory and produced thousands upon thousands of fertilized eggs. After carefully hatching and maintaining for almost 1 year, we successfully obtained about 500 first generation (F1) adults of B. belcheri and 3,300 of B. japonicum. Part of those F1 lancelets ripened and spontaneously spawned in 2006, and several thousands of second generation (F2) individuals of both species were produced. The young F2 lancelets are growing in good condition and some of B. japonicum initiated gonad development in December, 2006. Our experience emphasizes that cleanness of settlement substratum and sufficient food supply are important factors for long-term culture of lancelets in the laboratory.     

Biology of the amphioxus,Branchiostoma belcheri in the Ariake Sea,Japan I. Population structure and growth

We investigated the population structure and growth of the amphioxus Branchiostoma belcheri for four years in the southern Ariake Sea, Japan. We counted 62-66 myotomes and 251-310 dorsal fin-ray chambers, and these results support that this species is an intermediate form of B. belcheri and its subspecies B. belcheri tsingtauense. The ratio of females to males was 1:1.12. Males were more numerous than females among small individuals (< 40 mm body length), but we found no significant differences among large animals (50 mm body length). Spawning occurred from mid June to early July. Groups of newly settled young appeared from January to June of their second year. We observed a large fluctuation between years in the numbers of newly settled young. The estimated size of one-year-old individuals was 19.4 mm in body length; within the next 12 months, they reached 32.1 mm. Three- and four-year-old individuals measured 38.6 mm and 45.8 mm, respectively. Few grew beyond 60 mm; the largest specimen collected was a 64 mm male.     

Cloning and phylogenetic analysis of an amphioxus myogenic bHLH gene AmphiMDF

In this study, a member of the MyoD gene family, AmphiMDF, was isolated from the embryos of amphioxus by degenerate PCR, followed by rapid amplification of cDNA ends (RACE). Southern blot analysis confirmed that only a single myogenic bHLH gene was present in the genome of amphioxus Branchiostoma belcheri tsingtauense. Sequence and phylogenetic analyses indicated that AmphiMDF falls at the base of its vertebrate homologs. The amino acid sequence of AmphiMDF was almost equally similar to those of the four clusters of the vertebrate MyoD family. This suggests that AmphiMDF is not only the sister but also the archetype of the vertebrate myogenic bHLH genes. The scenarios to explain the origin of the vertebrate MyoD gene family from the ancestral myogenic bHLH gene like AmphiMDF are also discussed.     

雌雄文昌鱼同工酶的表型差异

本文应用聚丙烯酰胺凝胶电泳结合生化染色方法分析了雌雄文昌鱼中苹果酸酶、苹果酸脱氢酶、酸性磷酸酶和酯酶四种同工酶的酶谱。首次发现苹果酸酶、苹果酸脱氢酶和酸性磷酸酶表型在文昌鱼雌性和雄性个体之间存在差异 ,而在同一性别不同个体之间无差异。酯酶表型较复杂 ,不但在不同性别个体之间而且在同一性别不同个体之间都出现一定差异