Cloning and Comparative Studies of Seaweed Trehalose-6-Phosphate Synthase Genes

The full-length cDNA sequence (3219 base pairs) of the trehalose-6-phosphate synthase gene of Porphyra yezoensis (PyTPS) was isolated by RACE-PCR and deposited in GenBank (NCBI) with the accession number AY729671. PyTPS encodes a protein of 908 amino acids before a stop codon, and has a calculated molecular mass of 101,591 Daltons. The PyTPS protein consists of a TPS domain in the N-terminus and a putative TPP domain at the C-terminus. Homology alignment for PyTPS and the TPS proteins from bacteria, yeast and higher plants indicated that the most closely related sequences to PyTPS were those from higher plants (OsTPS and AtTPS5), whereas the most distant sequence to PyTPS was from bacteria (EcOtsAB). Based on the identified sequence of the PyTPS gene, PCR primers were designed and used to amplify the TPS genes from nine other seaweed species. Sequences of the nine obtained TPS genes were deposited in GenBank (NCBI). All 10 TPS genes encoded peptides of 908 amino acids and the sequences were highly conserved both in nucleotide composition (>94%) and in amino acid composition (>96%). Unlike the TPS genes from some other plants, there was no intron in any of the 10 isolated seaweed TPS genes.

海藻糖提高酵母拮抗菌生活力和生防效力的作用机制

近年来，利用酵母拮抗菌进行果实采后病害的生物防治已经成为果实采后领域的研究热点。但是，在实际应用中生物拮抗菌制剂的防病效果远不如化学药剂稳定。由于生物拮抗菌是活体，生活力和生防效力易受诸多因素影响。在商品化制剂的剂型加工、销售、以及使用过程中的环境条件往往影响酵母拮抗菌的生活力和抑病能力，成为酵母拮抗菌产业化生产和商品化应用过程中的一个主要障碍。将酵母拮抗菌和其它化学物质配合使用可以提高拮抗菌的生防效力。另外，增加酵母拮抗菌对逆境条件的耐受力也是增加或稳定其防治效果的有效途径。本文研究了海藻糖与酵母拮抗菌生活力和生防效力的关系，通过生理手段提高了酵母拮抗菌内源海藻糖的含量，同时探讨了在多种逆境条件下内源海藻糖含量对酵母拮抗菌生活力和生防效力的影响及其作用机制。主要研究结果如下： 1. 以1 %海藻糖作为碳源培养酵母拮抗菌Cryptococcus laurentii可以提高其内源海藻糖含量。在in vitro试验中，提高内源海藻糖含量可以提高C. laurentii在低温（1 ºC）、气调（1 ºC，5 % O2，5 % CO2）条件下的生活力;在in vivo试验中，提高C. laurentii内源海藻糖含量可以提高其在苹果果实伤口上的种群密度和对苹果青霉病的防治效果。内源海藻糖的积累还能提高C. laurentii冷冻干燥后的生活力，海藻糖对酵母细胞质膜的保护作用可能是一个主要原因。 2. 以1 %海藻糖作为碳源能提高酵母拮抗菌Rhodotorula glutinis的内源海藻糖含量。内源海藻糖含量的增加可以提高C. laurentii和R. glutinis在慢速冷冻处理中的生活力。同时，海藻糖作为外源保护剂可以明显提高两种酵母拮抗菌在冷冻干燥处理后的生活力。在快速冷冻、慢速冷冻和冷冻干燥处理中，提高酵母拮抗菌的内源海藻糖含量并使用海藻糖作为外源保护剂可以获得更高的生活力。同时，这种内、外源保护因子的综合作用也可以提高两种拮抗菌在苹果果实伤口上的种群密度和对苹果青霉病的防治效果。 3. 脱脂牛奶和糖（葡萄糖，半乳糖，蔗糖，海藻糖）作为保护剂可以提高C. laurentii冷冻干燥后在常温（25 ºC）和低温（4 ºC）保存过程中的生活力。脱脂牛奶和糖保护剂的复合使用对C. laurentii的保护效果高于其单独使用的效果。通过对几种常用的碳源进行筛选，发现柠檬酸作为碳源对C. laurentii内源海藻糖的积累有明显的诱导作用。当使用相同的保护剂或保护剂组合时，高内源海藻糖含量的酵母拮抗菌生活力更强。当冷冻干燥前使用半乳糖 + 脱脂牛奶作为保护剂时，高内源海藻糖含量的C. laurentii在4 ºC保存90 天后对苹果青霉病的防治效果和与新鲜培养的酵母拮抗菌相当。 4. 酵母拮抗菌C. laurentii冷冻干燥后在常温(25 ºC)保存期间，细胞生活力下降，细胞膜的完整性降低，胞内活性氧水平增加，同时与抗氧化相关的超氧化物歧化酶（SOD）活性也增加，而过氧化氢酶（CAT）活性则下降。提高内源海藻糖含量和/或使用外源保护剂（5 %脱脂牛奶 + 10 %葡萄糖）可以减缓上述指标下降或上升的速度。内、外源因子的共同作用有利于提高对拮抗菌细胞的保护作用。 5. 利用褐藻酸钠制成的含酵母拮抗菌的胶球在干燥后能有效的保持C. laurentii的生活力。在3种不同粘度的褐藻酸钠中，0.5 % 3500 cp的褐藻酸钠表现出较好的保护效果。内源海藻糖的积累可以提高干胶球中C. laurentii的生活力，作为外源保护剂的4种糖（葡萄糖、半乳糖、蔗糖、海藻糖）中只有海藻糖在低温条件下可以提高C. laurentii的生活力，其它3种糖反而降低了C. laurentii的生活力。

不同防冻剂对兔胚胎干细胞慢速冷冻保存的影响

干细胞冷冻保存是干细胞研究和临床应用中的必需技术.为提高兔胚胎干细胞在慢速冻存过程中的保存效果,比较了二甲基亚砜(DMSO)和乙二醇(ethylene glycol,EG)对兔胚胎干细胞冷冻保护效果.对冷冻复苏后的细胞进行台盼蓝染色,并研究其胚胎干细胞分子特性,结果表明DMSO比EG具有更好的冷冻保护效果.再在以10% DMSO为基础的防冻液中添加膜稳定剂海藻糖(trehalose)或谷氨酰胺(glutamine),细胞冷冻复苏后结果显示,谷氨酰胺对兔胚胎干细胞有明显的冷冻保护作用,使细胞存活率从71%提高到83.7%.当谷氨酰胺浓度为0、5、10、20、40 mmol/L分别加入防冻液中后,20 mmol/L的谷氨酰胺具有最佳的冷冻保护效果.以上结果得出兔胚胎干细胞慢速冷冻的防冻液改进配方为:在胚胎干细胞培养液中添加10% DMSO+20 mmol/L谷氨酰胺.

Vitrification of Yunnan Yellow Cattle oocytes: work in progress

The objective of this study was to provide a simple cryopreservation method for oocytes from Yunnan Yellow Cattle and facilitate preservation efforts in this native Chinese breed, which is threatened by agricultural modernization. Cumulus-oocyte complexes (COCs) were collected from slaughterhouse ovaries and matured in vitro for 22-24 h, then selected for cryopreservation. Vitrification in open pulled straws (OPS) or in microdrops on a cooled metal surface (solid surface vitrification, SSV) was compared. The OPS vitrification solution consisted of 20% ethylene glycol (EG) and 20% DMSO. The SSV solution was a mixture of 35% EG, 5% polyvinyl-pyrrolidon (PVP) and 0.4 M trehalose. Vitrified and warmed oocytes were either fertilized in vitro or parthenogenetically activated. The rates of cleavage and development to blastocysts of fertilized oocytes following OPS versus SSV were not statistically different (38.3 and 12.5% versus 35.8 and 6.0%, respectively). The corresponding rates of parthenogenetic development to blastocysts were also not different (8.2 versus 3.5%, respectively). Development to blastocysts of non-vitrified controls following fertilization was significantly higher than that of the vitrified oocytes (22.6%, P < 0.05). These results demonstrate for the first time, that although both OPS and SSV procedures reduced embryonic development, Yunnan Yellow Cattle oocytes are capable of developing to blastocysts following cryopreservation. (C) 2002 Elsevier Science Inc. All rights reserved.

Construction and characterization of a bacterial artificial chromosome library of marine macroalga Porphyra yezoensis (Rhodophyta)

A large-DNA-fragment library is necessary for research into the Porphyra genome. In this study, a bacterial artificial chromosome (BAC) library of Porphyra yezoensis was constructed and characterized. The library contains 54,144 BAC clones with an average insert size of about 65 kb and fewer than 0.7% of clones without large inserts. Therefore, its capacity is more than 6.6 P. yezoensis genome equivalents, and the probability of recovering any nuclear DNA sequence from the library is higher than 99%. The library shows good fidelity and stability. A putative trehalose-6-phosphate synthase (TPS) gene was successfully screened out from the library. The above results show that the library is useful for gene cloning and genomic research in P. yezoensis.

Discovery of the genes in response to white spot syndrome virus (WSSV) infection in Fenneropenaeus chinensis through cDNA microarray

We used microarray technology to study differentially expressed genes in white spot syndrome virus (WSSV)-infected shrimp. A total of 3136 cDNA targets, including 1578 unique genes from a cephalothorax cDNA library and 1536 cDNA clones from reverse and forward suppression subtractive hybridization (SSH) libraries of Fenneropenaeus chinensis, plus 14 negative and 8 blank control clones, were spotted onto a 18 x 18 mm area of NH2-modified glass slides. Gene expression patterns in the cephalothorax of shrimp at 6 h after WSSV injection and moribund shrimp naturally infected by WSSV were analyzed. A total of 105 elements on the arrays showed a similar regulation pattern in artificially infected shrimp and naturally infected moribund shrimp; parts of the results were confirmed by semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR). The up-regulated expression of immune-related genes, including heat shock proteins (HSP70 and HSP90), trehalose-phosphate synthase (TPS), ubiquitin C, and so forth, were observed when shrimp were challenged with WSSV. Genes including myosin LC2, ATP synthase A chain, and arginine kinase were found to be down-regulated after WSSV infection. The expression of housekeeping genes such as actin, elongation factor, and tubulin is not stable, and so these genes are not suitable as internal standards for semiquantitative RT-PCR when shrimp are challenged by WSSV. As a substitute, we found that triosephosphate isomerase (TPI) was an ideal candidate of interstandards in this situation.