A transglutaminase from Chinese shrimp (Fenneropenaeus chinensis), full-length cDNA cloning, tissue localization and expression profile after challenge

Transglutaminase can catalyze the cross-linking reaction between soluble clotting protein molecules from the plasma for prevention of excess blood loss from a wound and obstructing micro-organisms from invading the wound in crustaceans. A novel transglutaminase (FcTG) gene was cloned from hemocytes of Chinese shrimp Fenneropenaeus chinensis by 3' and 5' rapid amplification of cDNA ends (RACE) PCR. The full-length cDNA consists of 2972 bp, encoding 757 amino acids with a calculated molecular mass of 84.96 kDa and a theoretical isoelectric point of 5.61. FcTG contains a typical transglutaminase-like homologue (TGc domain: E-value = 1.94e-38). Three catalytic sites (Cys-324, His-391 and Asp-414) are present in this domain. The deduced amino acid sequence of FcTG showed high identity with black tiger shrimp TG, kuruma shrimp TG and crayfish TG. Transcripts of FcTG mRNA were mainly detected in gill, lymphoid organ and hemocytes by RT-PCR. RNA in situ hybridization further confirmed that FcTG was constitutively expressed in hemocytes both in the circulatory system and lymphoid organ. The variation of mRNA transcription level in hemocytes and lymphoid organ following injection of killed bacteria or infection with white spot syndrome virus (WSSV) was quantified by RT-PCR. The up-regulated expression of FcTG in shrimp lymphoid organ following injection of bacteria indicates that it is inducible and might be associated with bacterial challenge. (c) 2006 Elsevier Ltd. All rights reserved.

Molecular cloning and characterization of a thioester-containing protein from Zhikong scallop Chlamys farreri

Thioester-containing proteins are a family of proteins characterized by the unique intrachain beta-cysteinyl-gamma-glutamyl thioester, which play important roles in innate immune responses. The cDNA of Zhikong scallop Chlamys farreri thioester-containing protein (designated as CfTEP) was cloned by expressed sequence tag (EST) and rapid amplification of cDNA ends (RACE) approaches. The full-length cDNA of CfTEP was of 4616 bp, consisting of a 5 '-terminal untranslated region (UTR) of 30 bp and a 3 ' UTR of 140 bp with a polyadenylation signal sequence AATAAA and a poly(A) tail. The CfTEP cDNA encoded a polypeptide of 1481 amino acids with the theoretical isoelectric point of 5.98 and the predicted molecular weight of 161.4 kDa. The deduced amino acid sequence of CfTEP contained the canonical thioester motif GCGEQ, nine potential N-glycosylation sites and a C-terminal distinctive cysteine signature. It also contained a presumed catalytic histidine and proteolytic cleavage sites that were similar to C3 molecules. The high similarity of CfTEP with the thioester-containing proteins in other organisms, such as the TEPs from insects, the complement component C3, C4, C5 and the protease inhibitor alpha(2)-macroglobulin indicated that CfTEP should be a member of TEP family. The phylogenetic analysis revealed that CfTEP was closely related to TEPs from mollusc, nematodes and insects, and they formed a separate branch apart from the branches of complements factors and alpha(2)-macroglobulins. The spatial expression of CfTEP transcripts in healthy and bacterial challenged scallops was examined by semi-quantitative RT-PCR. The CfTEP transcripts were mainly detected in the tissues of hepatopancreas and gonad, and remarkably up-regulated by Microbial challenge, which suggested that CfTEP was a constitutive and inducible acute-phase protein involved in immune defense. These results provided new insights into the role of CfTEP in scallop immune responses, as well as the evolutionary origin of this important, widespread and functionally diversified family of proteins. (c) 2007 Published by Elsevier Ltd.

QTL Mapping for Frond Length and Width in Laminaria japonica Aresch (Laminarales, Phaeophyta) Using AFLP and SSR Markers

In Laminaria japonica Aresch breeding practice, two quantitative traits, frond length (FL) and frond width (FW), are the most important phenotypic selection index. In order to increase the breeding efficiency by integrating phenotypic selection and marker-assisted selection, the first set of QTL controlling the two traits were determined in F-2 family using amplified fragment length polymorphism (AFLP) and simple sequence repeat (SSR) markers. Two prominent L. japonicas inbred lines, one with "broad and thin blade" characteristics and another with "long and narrow blade" characteristics, were applied in the hybridization to yield the F-2 mapping population with 92 individuals. A total of 287 AFLP markers and 11 SSR markers were used to construct a L. japonica genetic map. The yielded map was consisted of 28 linkage groups (LG) named LG1 to LG28, spanning 1,811.1 cM with an average interval of 6.7 cM and covering the 82.8% of the estimated genome 2,186.7 cM. While three genome-wide significant QTL were detected on LG1 (two QTL) and LG4 for "FL," explaining in total 42.36% of the phenotypic variance, two QTL were identified on LG3 and LG5 for the trait "FW," accounting for the total of 36.39% of the phenotypic variance. The gene action of these QTL was additive and partially dominant. The yielded linkage map and the detected QTL can provide a tool for further genetic analysis of two traits and be potential for maker-assisted selection in L. japonica breeding.

Genetic mapping of laminaria japonica and l. longissima using amplified fragment length polymorphism markers in a "two-way pseudo-testcross" strategy

With a "two-way pseudo-testcross" mapping strategy, we applied the amplified fragment length polymorphism (AFLP) markers to construct two moderate density genetic linkage maps for Laminaria. The linkage maps were generated from the 60 progenies of the F, cross family (Laminaria longissima Aresch. x L. japonica Miyabe) with twenty pairs of primer combinations. Of the 333 polymorphic loci scored in 60 progenies, 173 segregated in a 1:1 ratio, corresponding to DNA polymorphisms heterozygous in a single parent, and the other 58 loci existing in both parents followed a 3:1 Mendelian segregation ratio. Among the loci with 1:1 segregating ratios, 79 loci were ordered in 14 linkage groups (648.6 cM) of the paternal map, and 72 loci were ordered in 14 linkage groups (601.9 cM) of the maternal map. The average density of loci was approximately 1 per 8 cM. To investigate the homologies between two parental maps, we used 58 loci segregated 3:1 for further analysis, and deduced one homologous linkage group. The linkage data developed in these maps will be useful for detecting loci-controlling commercially important traits for Laminaria.

Identification and mapping of amplified fragment length polymorphism markers linked to shell color in bay scallop, Argopecten irradians irradians (Lamarck, 1819)

Amplified fragment length polymorphisms (AFLP) were used to study the inheritance of shell color in Argopecten irradians. Two scallops, one with orange and the other with white shells, were used as parents to produce four F-1 families by selfing and outcrossing. Eighty-eight progeny, 37 orange and 51 white, were randomly selected from one of the families for segregation and mapping analysis with AFLP and microsatellite markers. Twenty-five AFLP primer pairs were screened, yielding 1138 fragments, among which 148 (13.0%) were polymorphic in two parents and segregated in progeny. Six AFLP markers showed significant (P < 0.05) association with shell color. All six loci were mapped to one linkage group. One of the markers, F1f335, is completely linked to the gene for orange shell, which we designated as Orange1, without any recombination in the progeny we sampled. The marker was amplified in the orange parent and all orange progeny, but absent in the white parent and all the white progeny. The close linkage between F1f335 and Orange1 was validated using bulk segregation analysis in two natural populations, and all our data indicate that F1f335 is specific for the shell color gene, Orange1. The genomic mapping of a shell color gene in bay scallop improves our understanding of shell color inheritance and may contribute to the breeding of molluscs with desired shell colors.

Its length polymorphism in oysters and its use in species identification

In an effort to develop genetic markers for oyster identification, we studied length polymorphism in internal transcribed spacers (ITS) between major ribosomal RNA genes in 12 common species of Ostreidae: Crassostrea virginica, C. rhizophorae, C. gigas, C. angulata, C. sikamea, C. ariakensis, C. hongkongensis, Saccostrea echinata, S. glomerata, Ostrea angasi, O. edulis, and O. conchaphila. We designed two pairs of primers and optimized PCR conditions for simultaneous amplification of ITS 1 and ITS2 in a single PCR. Amplification was successful in all 12 species, and PCR products were visualized on high-resolution agarose gels. ITS2 was longer than ITS 1 in all Crassostrea and Saccostrea species, whereas they were about the same size in the three Ostrea species. No intraspecific variation in ITS length was detected. Among species, the length of ITS I and ITS2 was polymorphic and provided unique identification of 8 species or species pairs: C. ariakensis, C. hongkongensis, C. sikamea, O. conchaphila, C. virginica/C. rhizophorae, C. gigas/C. angulata, S. echinata/S. glonzerata, and O. angasi/O. edulis. The ITS assay provides simple, rapid and effective identification of C. ariakensis and several other oyster species. Because the primer sequences are conserved, the ITS assay may be useful in the identification of other bivalve species.

Stable Carbon Isotopic Composition in Soil Organic Carbon and C3/C4 Source Variations at the Haibei Alpine Meadow

Stable carbon isotopes of organic matter originated from different soil layers (0～5 cm, 5～15 cm, 15～25 cm, 25～35 cm, 35～50 cm, 50～65 cm) were investigated in the Haibei Alpine Meadow Ecosystem Research Station of the Chinese Academy of Sciences. The preliminary results indicated that δ13C values of soil organic matter increased with increased soil depth. δ13C of soil organic carbon in 0～5 cm layer showed the lowest value, -25.09‰; while 50～65 cm soil layer possessed the lowerδ13C value, -13.87‰. Based on mass balance model of stable isotopes, it was proposed that the percentage of C4 carbon source tend to increase with increased soil depth. The preliminary study indicated that alpine meadow might have undergone a successive process from C4-dominated community to C3-dominated one. However, changing δ13C values in atmospheric CO2 overtime and different processes of soil organic carbon formation (or eluviation) might somewhat contribute to increasing δ13C values. In this case, mass balance model would underestimate C3 community and overestimate C4 community.

Stable carbon isotope analyses show dominance of C3 grasses in the feed selected by small herbivores at the alpine meadow at the Tibetan Plateau

Muscle samples were collected from small herbivorous mammals (Ochotona curzoniae, Microtus oecnomus, Myospalax fontanierii and Lepus oiostolus) at the alpine meadow ecosystem at the Tibetan Plateau in order to address variability in stable carbon isotope composition. Stable carbon isotope values of muscles remain steady and show no significant variations (-25.72 to -27.04 parts per thousand) among the four small mammal species. Based on the mass balance theory of stable isotopes, it is proposed that small herbivorous mammals mainly (or totally) rely on C3 grasses as food supply, and there is few or no distribution of C4 grasses at the ecosystem. The results reflect our previous study on the isotope patterns of plant species. Thus, stable carbon isotope analysis of muscles provides a method to address dietary selection and dietary variability in herbivores. In addition, stable carbon isotopic analyses can be used to address changes in vegetation distributions in ecosystem and paleovegetaion and paleoclimate.

北天山新生代构造演化与古环境演变

The Tianshan Mountains is located about 1000-2000 km north of the India-Asia suture and is the most outstanding topography in central Asia, with transmeridional length of nearly 2500 km, north-southern wideness of ~ 300-500 km, peaks exceeding 7000 m above sea level (asl.), and average altitude of over 4000 m asl. Much of the modern relief of the Tianshan Range is a result of contraction driven by the collision of the India subcontinent with the southern margin of Asia, which began in early Tertiary and continues today. Understanding where, when and how the deformation of the Tianshan Mountains occurred is essential to decipher the mechanism of intracontinental tectonics, the process of foreland basin evolution and mountain building, and the history of climate change in central Asia. In order to better constrain the Cenozoic building history of the Tianshan Mountains and the climate change in the southern margin of the Junggar Basin, we carried out multiple studies of magnetostratigraphy, sedimentology, and stable isotopes of paleosol carbonate at the Jingou River section, which is located at the Huoerguosi anticline, the westernest one of the second folds and thrust faults zone in the northern piedmont of the Tianshan Mountains. The Jingou River section with a thickness of about 4160 m is continuous in deposits according to the observed gradual change in sedimentary environments and can be divided into five formations: Anjihaihe, Shawan, Taxihe, Dushanzi and Xiyu in upward sequence. Characteristic remamences were isolated by progressive thermal demagnetization, generally between 300 and 680℃. A total of 1133 out of 1607 samples yielded well-defined ChRMs and were used to establish the magnetostratigraphic column of a 3270-m-thick section from the exposed base of the Anjihaihe Formation to the middle of the Xiyu Formation. Two vertebrate fossil sites and a good correlation with the CK95 geomagnetic polarity time scale suggest that the section was deposited from ~30.5 to ~4.6 Ma and the age of the top of the Xiyu formation is ~2.6 Ma based on an extrapolation of the sedimentation rates. A plot of magnetostratigraphic age vs. height at the Jingou River section shows that significant increases in sedimentation rates as well as notable changes in depositional environments occurred at ~26-22.5 Ma, ~13-11 Ma and ~7 Ma, which represent the initial uplift of the Tianshan Mountains and two subsequent rapid uplift events. In addition, changes in sedimentation rates display characteristic alternations between increases and decreases, which probably indicate that the uplift of the Tianshan Mountains was episodic. We discussed the history of C4 biomass and climatic conditions in the southern margin of the Junggur Basin using the stable carbon and oxygen isotope composition of paleosol carbonates from the Jingou River section during ~17.5-6.5 Ma. The δ13C values indicate that the proportion of C4 biomass was uniform and moderate (15-20 %) during the interval of ~17.5-6.5 Ma. We proposed three hypotheses for this pattern of C4 biomass: (1) counteraction of two opposed factors (global cooling since ~15 Ma and thereafter increased dry and seasonality in central Asia) controlling the growth of C4 grasses, (2) variability in abundance of C3 grasses relative to C3 trees and shrubs if vegetation had ever changed in ecosystems, and (3) the higher latitude of the studied region. The δ18O values show a stepwise negative trend since ~13 Ma which may be attributed to three factors: (1) the temperature decreasing gradually after the middle Miocene (~15 Ma), (2) the increasing contribution of the moistures carried by the polar air masses from the Arctic Ocean to precipitation, and (3) the gradual retreat westward and disappearance of the Paratethys Ocean. Among them, which one played a more important role will need further study of the paleoclimate in central Asia.

陆生蜗牛壳体同位素组成及其对生长季节的响应

Stable isotope compositions of land snail shells have a great potential as an indicator of paleoclimatic and paleoenvironmental changes. However, some key issues, such as the relationship of carbon isotope between snail food and local vegetation, and the uncertainty of the dominant factors about snail body fluid changes in oxygen isotope composition, remain less well known, strongly limiting shell isotopic application. In this study, we measure the stable isotope compositions on the shells of both live snails and fossils collected from the Chinese Loess Plateau and a loess sequence at Mangshan, Xingyang, respectively. Based on the analyses, the association of the stable isotope compositions of land snail shells with their growing seasons is investigated. In addition, the climatic and environmental significances of isotopic differences among several snail species are discussed. The main results and conclusions are presented as follows: 1. δ18O values for the shell lip samples of Bradybaena ravida redfieldi range from -6.79‰ to -1.92‰, and parallels to the monthly changes of local rain water δ18O, temperature and humidity. The compatibility of shell lip δ18O with monthly modeled shell δ18O indicates that the shell lip δ18O changes are mainly resulted from the 18O variations of rain-water. The shells of a land snail growing in spring could be enriched in 18O, and those growing in summer depleted in 18O. 2. Carbon isotope compositions of snail shells are controlled by their diet, which is affected by the relative proportion of C3 to C4. There are some differences in carbon isotopic compositions among different snail species, especially between P. orphana and V. tenera or P. aeoli. Shell δ13C for P. orphana is the most positive with an average of -5.88 ± 2.54 ‰. The C4 plant fraction of the food for “cold-aridiphilous” taxa, P. aeoli and V. tenera, is distinctly lower than that for “thermo-humidiphilous” taxa, P. orphana, indicating that summer is likely to be the main active season of P. orphana and spring of P. aeoli and V. tenera. Therefore, some discrepancy of carbon isotopic compositions among different species may be related to snail active season. 3. δ13C values among different species have a certain degree of positive correlation, which may be influenced by local vegetation ecosystem. δ13C value of the snail shells (especially P. orphana) shows an eastward increasing trend and consists with the variations of C4 plants biomass in Loess Plateau. The result shows that the carbon isotope in local vegetation ecosystem is one of the main factors influencing δ13C values of snail food. Therefore, both carbon isotopes of local vegetation ecosystem and snail active season contribute to the carbon isotopic differences among different snail species and in different areas. 4. δ13C values of living snail shells and soil organic matter have a positive correlation with each other, which further supports the view that carbon isotope in local vegetation ecosystem is one of the main factors influencing δ13C values of snail food. However, the range of δ13C values of snail food for various species in response to carbon isotope in local vegetation ecosystem is different. It is suggested that 13C enrichment of snail shells relative to local vegetation ecosystem has a potential to indicate snail active season and the degree of climate temperature and humidity. 5. There is a significant negative correlation between carbon and oxygen isotopic compositions of living snail shells in Loess Plateau. This result further supports that snail active season can be inferred based on the shell carbon and oxygen isotopic compositions. Moreover, there are some positive correlations between mean annual temperature and differences of shell δ13C values ( 13CV. tenera-P. orphana) and that of δ18O values ( 18OV. tenera-P. orphana) for P. orphana, a typical “thermo-humidiphilous” taxa, and V. tenera, a typical “cold-aridiphilous” taxa, respectively. It shows that  13CV. tenera-P. orphana and  18OV. tenera-P. orphana may have a potential to indicate mean annual temperature or the length of biological growing season. 6. Stable isotopes of land snail shell in the Mangshan loess sequence show that the shell δ18O value of “cold-aridiphilous” taxa V. tenera is more positive than “thermo-humidiphilous” taxa P. orphana and δ13C value of the former is more negative than the latter. In addition, the shell δ18O value of V. tenera varies significantly in different period. During the last glacial maximum, its δ18O value with an average of -7.89 ‰ is more negative than that (-5.88 ‰) from the last deglaciation to the early Holocene. This phenomenon indicates that its growing season during different period is significantly different. It tends to grow in summer in last glacial maximum. With climate warming, it prefers growing in spring with relatively low temperature. While the shell δ18O value of P. orphana varies in a little range, which shows that its activity season is shorter and mainly in summer. These results further support that the change of the snail growing season is one of the main factors of differences of carbon isotopic compositions among different snail species and varies with time. Furthermore, it is consistent that changes in magnetic susceptibility and trend of differences of shell δ18O values and δ13C values respectively between the two snail fossils. It is further testified that 13CV. tenera-P. orphana and  18OV. tenera-P. orphana may have a potential to indicate mean annual temperature or the length of biological growing season.

贵州石灰岩和砂岩地区C4和C3植物营养元素的化学计量对N／P比值波动的影响

陆地生态系统植物的生长受到营养元素氮（N）和磷（P）的可利用性的限制。已有的证据表明营养元素的相对丰度将控制生态系统的营养元素循环和能量流动的速度。文章提出如下假设：为了适应环境的变化，植物具有可伸缩性地调整营养元素含量的能力，也就是营养元素化学计量比值变化的能力，植物N／P比值波动的影响不仅来源于N对P的相对可利用性的变化，也来源于其他营养元素化学计量的变化，尤其是与Ca的化学计量的变化。为了验证上述假设，本研究利用3种C4植物和11种C3植物，研究了植物N／P化学计量比值的波动随N与Ca和P与Ca化学计量的变化模式：对C4植物来说，N／P比值的波动主要受生物量P与Ca化学计量变化的影响；而对C3植物来说，则同时受N与Ca和P与Ca化学计量变化的控制，它们之间的相对控制能力的大小将决定植物N／P比值波动的变化梯度，C4植物和C3植物的N／P比值的波动都要受土壤pH值的影响。本研究对了解物种丰度和N对P的相对可利用性、N与Ca，以及P与Ca的化学计量之间关系具有重要意义.