牛耳草Bose hygrometrica 离体叶脱水-复水过程的光合作用及基因表达

本文以复苏植物牛耳草Boea hygrometrica成熟植株的离体叶片为试材，对比非复苏植物烟叶唇苣苔Chirita heterotricha, 以光合作用在脱水－复水过程中的变化为切入点，从生理水平上探讨其脱水保护位点：应用mRNA差异显示技术，从分子水平上探讨其脱水保护机制。 光合放氧速率、快速荧光诱导动力学、慢速荧光诱导动力学、荧光发射光谱、荧光激发谱的结果表明，相对于烟叶唇柱苣苔，脱水对牛耳草净光合速率、PS II和PS I光化学活性、电子传递、光合磷酸化及CO_2固定的影响有一个共同的特点，即脱水时迅速降低，复水后恢复能力强。通过非变性绿胶的研究牛耳草叶片类囊体膜叶绿素－蛋白复合体在脱水－复水过程中保持高度稳定。色素含量分析表明牛耳草的叶绿素含量在脱水－复水过程中也相对稳定。这些特征可能是牛耳草叶片光合作用脱水保护机制的一部分。 SDS－PAGE和IEF电泳结果表明，牛耳草脱水复苏过程中蛋白质表达有差异，或增或减，并分别发现了一条（SDS－PAGE）和两条（IEF）在脱水过程中特异出现的蛋白质。 本文以银染法代替放射自显影用于mRNA差异显示，不但简化了实验步骤，缩短了实验周期，而且在不降低灵敏度的前提下避免了放射性危害，降低了实验成本。本文证明了mRNA差异银染显示法用于复苏植物牛耳草脱水－复水过程中基因表达变化的研究是可行的。 mRNA差异银染显示法揭示牛耳草耐脱水复苏机制涉及到基因表达的调控。脱水－复水过程中差异表达的基因有6种，其中脱水特异诱导表达的13个cDNA所相应的基因、脱水上调节的15个cDNA所相应的基因可能参与牛耳草叶片脱水保护机制，复水特异诱导的8个cDNA的所相应基因可能参与牛耳草复水后的修复机制。2个脱水特异诱导表达的cDNA片段进行了克隆和测序。

拟南芥AST基因的精细作图

拟南芥ast (anthocyanin spotted testa) 突变体是由碳离子束诱导产生的与花青苷生物合成有关的突变体，受单隐性核基因控制。由于花青苷的异常积累，突变体未成熟种子的种皮呈现紫红色的斑点;野生型植株幼嫩的种皮没有花青苷的异常积累，呈淡绿色。初步作图分析表明，AST基因定位于拟南芥第I号染色体上，并且位于SSLP分子标记nga280和CAPS分子标记PAB5之间。 AST基因与SSLP分子标记nga280紧密连锁，遗传距离为3.2cM;与CAPS分子标记PAB5相距较远，遗传距离为21.1cM。 采用DDRT-PCR的策略，分析野生型与突变型植株未成熟角果中基因表达的差异。通过调整DDRT-PCR中总RNA、锚定引物、随机引物、cDNA和dNTP等关键试剂的用量，优化了适用于银染检测的DDRT-PCR方法。PCR扩增产物经6%变性聚丙烯酰胺凝胶垂直电泳分离后，银染能检测到多而清晰的条带。泳道中的条带数最少为40个，最多达80个，平均为60个，条带大小分布在100bp-900bp范围，银染的灵敏度为5pg/mm2。此方法操作简便快速，灵敏度高，重复性好。采用这个改良的的方法，分析了拟南芥野生型和ast突变型植株未成熟角果中16,000个cDNA扩增产物条带，从中筛选出28个差异条带。二次PCR扩增后，进一步筛选出10个差异表达的cDNA条带，其中6个是野生型特异表达的，4个是突变型特异表达的。对这10个差异片段进行测序。BLASTN分析表明，这10个差异表达的cDNA片段与数据库中花青苷生物合成途径中的结构基因和调节基因序列没有同源性，表明用DDRT-PCR的方法克隆特定的AST基因有一定的局限性。 利用图位克隆（map-based cloning）的策略，对拟南芥AST 基因进行克隆。根据拟南芥数据库中的SNPs (simple nucleotide polymophisms) 序列和插入/缺失多态性（insertion/deletion polymorphisms）序列，设计了一系列分子标记。利用这些分子标记，对600个F2代有突变表型的植株进行重组子筛选，完成了对拟南芥AST基因的精细作图，成功地将AST 基因定位到BAC克隆T13M11上。初步确定该BAC克隆中的基因T13M11.8 可能是AST基因。该基因的DNA序列长1432bp，含有6个外显子和5个内含子，编码的蛋白与花青苷生物合成途径中的二氢黄酮醇4-还原酶有较高的同源性。功能互补实验正在进行当中。

I.小麦非叶器官碳酸酐酶活性与定位分析II.镉毒害导致芦苇胚性细胞死亡的激素调控研究

第一部分： 通过生理测定和化学染色分析了冬小麦品种小堰54和京411的叶片和非叶片组织的碳酸酐 酶活性。叶片碳酸酐酶活性(CA)在挑旗时期达到最大值，之后减少到最小，而在饱粒期又呈 增加趋势。从灌浆期到饱粒期，颖片和内稃的CA活性均减少，而外稃和种皮的CA活性均增加。在饱粒期，小堰54的叶片、颖片、外稃和种皮CA活性均高于京411。组织化学染色表明，CA主要分布在旗叶的叶肉细胞叶绿体中，也分布在非叶片组织颖片、外稃和内稃的叶肉和维管束鞘细胞的胞质中。这些结果表明，小麦非叶组织叶肉和维管束鞘细胞的胞质中的CA可能对饱粒期冬小麦的C4光合途径起作用。饱粒期小堰54的C02传递到Rubisco酶速率和抗旱性较京411高。 第二部分： 以继代培养的芦苇胚性细胞为材料，利用台盼兰拒染法检测了悬浮细胞死亡过程，并利用石蜡切片法及苏木精染色法观察了不同浓度镉对芦苇细胞的毒害作用。1000μM的CdCl2迅速导致芦苇悬浮细胞死亡，200μM的CdClz在接种后第5天引起悬浮细胞死亡，100μM的CdCl2在接种后第7天引起悬浮细胞死亡，≤50μM的CdClz在接种后7天不引起悬浮细胞死亡。同时对不同浓度镉处理的芦苇胚性细胞的内源植物激素和可溶性蛋白质进行分析，≤50μM的镉浓度显著地降低胚性细胞内IAA、ZR、GA3和GA4的含量，却提高ABA的含量，抑制可溶性蛋白质的合成：≥100μM的镉浓度显著地提高IAA、ZR、GA3和GA4的含量，却降低ABA的含量，促进可溶性蛋白质的合成。这些结果表明，镉的毒害至少包括镉浓度决定的两种细胞死亡机制，高浓度的镉(1000μM)引起的细胞死亡应当为坏死，而100μM的镉引起植物悬浮细胞发生程序性死亡。在较高浓度(≥100μM)的镉处理下，芦苇细胞内内源IAA、ZR、GA3和GA4的浓度较高，可能调控可溶性蛋白质的合成而促进细胞发生程序化死亡。

MADS-box基因在单子叶植物花发育中的功能研究

第一部分 水稻E类MADS-box 基因在花发育中的功能分析 MADS-box 基因是一个大的转录因子家族，在花发育过程中起重要作用。根据对双子叶模式植物拟南芥、金鱼草和矮牵牛遗传突变体的研究，提出了花发育的ABCDE模型。该模型认为：A、B、C、D、E代表了5类功能不同的花器官特征基因，单独或联合控制花器官的发育。A类基因控制萼片的发育;A、B和E类基因控制花瓣的发育;B、C和E类基因控制雄蕊的发育;C和E类基因控制心皮的发育;D类基因控制胚珠的发育;A和C类基因相互抑制。在这5类基因中，E类基因的功能较为复杂，它不仅是花器官特征基因，而且具有花分生组织决定性（Floral meristem determinency）。在单子叶植物中，E类基因的功能发生了很大的分化。水稻是单子叶植物的模式植物，水稻中至少有5个E类基因，分别是OsMADS1、OsMADS5、OsMADS7、OsMADS8和OsMADS34，在这5个E类基因中，除了对OsMADS1基因有较深入的研究外，对其它几个E类基因的功能了解甚少。我们在现有的研究基础上，根据对双子叶植物中E类基因的研究结果，以OsMADS8基因为出发点，利用组织原位杂交，RNAi技术对水稻中的E类基因进行了深入的研究。结果表明：OsMADS8/7基因早在花序枝梗分生组织原基就有转录，随着小穗的生长发育，逐渐集中在小穗分生组织原基，小花分生组织原基，浆片、雄蕊和心皮中表达;在胚珠形成时，内外珠被有很强的杂交信号，而且在幼胚和胚乳中也有表达。OsMADS5在幼花时期，四轮花器官均有表达，在小穗发育后期及受精后的表达方式与OsMADS8/7基因相同。OsMADS8基因被抑制后，转基因植株没有任何表型变化，说明很可能有其它E类基因弥补了OsMADS8基因的功能缺失;当同时抑制其它E类基因的表达时，转基因植株抽穗期明显延长，四轮花器官的发育均受到影响：稃片类似叶片状;浆片转变为稃片类的结构;雄蕊没有花粉;心皮具有了稃片的特点;没有胚珠结构的形成，同时失去了花分生组织决定性，在心皮的部位产生了新的花器官或花分生组织逆转为花序分生组织。说明水稻四轮花器官及胚珠的正常发育需要E类基因的参与，但其功能与双子叶植物如拟南芥，西红柿、矮牵牛等直系同源基因相比已经发生变化;水稻中的E类基因在维持花分生组织特征性方面起重要作用;另外对抽穗期有影响。 第二部分 玉米MADS-box基因ZAG2转录调控区的研究 基因的时空表达受基因中的顺式作用元件及其反式作用因子调控。顺式作用元件由位于基因编码区上游的启动子区域和位置不确定的增强子区域组成。顺式作用元件对基因表达的开启至关重要。MADS-box 基因编码一类控制花器官发育的转录因子，在花的发育过程中顺序表达。MADS-box 基因突变，花器官发生同源异型转换。研究MADS-box 基因的调控序列可以进一步揭示影响基因时空表达的内外因素。ZAG2是玉米MADS-box 基因中的D类基因，控制胚珠的发育，在胚珠和心皮的内表面特异表达。ZAG2基因有7个外显子和6个内含子。我们从玉米基因组分离到了ZAG2基因翻译起始点上游3040bp的序列，并利用5’-RACE方法鉴定出了转录起始点的位置。序列比较发现，在 5’-UTR内有一个1299bp的内含子，这个内含子可能对基因的表达有调控作用，因此构建了两个与GUS基因融合的表达载体：一个是pZAG2-1::GUS，包括翻译起始点以上所有的调控序列;另一个是pZAG2-2::GUS，去掉了5’-UTR中的内含子序列，转化水稻。结果这两个构建都没有使GUS基因在正确的位置表达。pZAG2-1::GUS构建在心皮基部类似花托的部位及稃片顶端着色，pZAG2-2::GUS构建在内外稃片沿稃脉的部位有很强的着色，说明翻译起始点上游的调控序列不足以使基因正常表达。两个构建着色方式不同，可能pZAG2-1::GUS构建在5’-UTR部分含有抑制ZAG2基因在稃片表达的顺式元件，或者启用了在5’-UTR中的转录起始点，因为在5’-UTR的内含子中也有一个很典型的TATA-box。我们推测，在ZAG2基因编码区的第一内含子可能存在另外一些使基因正常表达的增强元件，需要进一步的序列缺失实验加以验证。

Saccharibacillus kuerlensis sp nov., isolated from a desert soil

A taxonomic study was performed on strain HR1(T), which was isolated from a desert soil sample collected from Xinjiang Province (China). Cells were aerobic, Gram-positive-staining, pink-pigmented, sporulating rods with a single lateral flagellum. The orga

Single UV excitation of Hoechst 33342 and propidium iodide for viability assessment of rhesus monkey spermatozoa using flow cytometry

Many fluorescent probes excited by visible light have been used to assess sperm quality by flow cytometry. Developing a viability evaluation method using UV excited stains would be useful for multiparameter analysis of sperm function. This investigation was conducted to determine the efficacy of Hoechst 33342 (H342) and propidium iodide (PI) dual staining for evaluating rhesus monkey sperm viability through use of flow cytometry and excited by a single UV laser. The results showed that the live cells stained only with H342 strongly correlated with expected sperm viability, and flow cytometric analyses were highly correlated with fluorescence microscopic observation. Using H342/PI/SYBR-14 triple staining method, it was found that the live/dead sperm distributions were completely concordant in both H342/PI and SYBR-14/PI assays. In addition, this dual staining was extended with fluorescein isothiocyanate-conjugated peanut agglutinin (FITC-PNA) to simultaneously analyze viability and acrosome integrity of sperm cryopreserved using two different extenders, TTE and TEST, and indicated that TTE offered better Preservation of plasma and acrosome integrity than TEST Therefore, the H342/PI dual staining provides an accurate technique for evaluating viability of rhesus monkey sperm and should be valuable for multiparameter flow cytometric analysis of sperm function.

KARYOTYPE AND BANDING-PATTERNS OF FEA TREE RAT (CHIROMYSCUS-CHIROPUS)

Fea's tree rat (Chiromyscus chiropus) is a very rare species which there are only a few specimens in the world. The chromosomes of two male specimens, collected from Xishuanbanna, Yunnan, are analysed by several banding technique (G-, C-bands, as well as Ag-staining). The diploid chromosome number is 22, and autosomes comprise 5 pairs of metacentrics, 2 pairs of subacrocentrics, and 3 pairs of acrocentrics. The X chromosome is a acrocentric, and Y is a micro-chromosome, almost a point, which could be a marker chromosome of the species and the genus. The centromeric C-bands are very faint, and C-bands of Nos. 1, 2, 9 and Y chromosome are negative. Only one pair Ag-NORs was found on No. 10 in the silver-stained karyotype. The relationship between morphologic and chromosomal features was discussed, and C-banded karyotype evolutionary trend has also been discussed. Moreover, the conventional karyotype of Niviventer confucianus was described.

HORMONAL-REGULATION OF TISSUE-TYPE PLASMINOGEN-ACTIVATOR AND PLASMINOGEN-ACTIVATOR INHIBITOR TYPE-1 IN CULTURED MONKEY SERTOLI CELLS

Sertoli cells play a central role in the control and maintenance of spermatogenesis. Isolated Sertoli cells of mouse and rat testes have been shown to secrete plasminogen activator (PA) and a plasminogen activator inhibitor type-1 (PAI-1) in culture. In this study, we have investigated the hormonal regulation of PA and PAI-1 activities in cultured monkey Sertoli cells. Sertoli cells (5x10(5) cells/well) isolated from infant rhesus monkey testes were preincubated at 35 degrees C for 16 h in 24-well plates precoated with poly(D-lysine) (5 mu g/cm(2)) in 0.5 mi McCoy's 5a medium containing 5% of fetal calf serum and further incubated for 48 h in 0.5 mi serum-free medium with or without various hormones or other compounds, PA as well as PAI-1 activities in the conditioned media were assayed by fibrin overlay and reverse fibrin autography techniques respectively. The Sertoli cells in vitro secreted only tissue-type PA (tPA), no detectable amount of urokinase-type PA (uPA) could be observed, Monkey Sertoli cells were also capable of secreting PAI-1, Immunocytochemical studies indicated that both tPA and PAI-1 positive staining localized in the Sertoli cells, spermatids and residual bodies of the seminiferous epithelium; Northern blot analysis further confirmed the presence of both tPA and PAI-1 mRNA in monkey Sertoli cells. Addition of follicle-stimulating hormone (FSH) or cyclic adenosine monophosphate (cAMP) derivatives or cAMP-generating agents and gonadotrophin-releasing hormone (GnRH) agonist or phorbol ester (PMA) to the cell culture significantly increased tPA activity. PAI-1 activity in the culture was also enhanced by these reagents except 8-bromo-dibutyryl-cAMP, forskolin and 3-isobutyl-1-methylxanthin (MIX) which greatly stimulated tPA activity, whereas decreased PAI-1 activity, implying that neutralization of PAI-1 activity by tile high level of tPA in the conditioned media may occur. These data suggest that increased intracellular signals which activate protein kinase A (PKA), or protein kinase C (PKC) can modulate Sertoli cell tPA and PAI-1 activities, The concomitant induction of PA and PAI-1 by the same reagents in the Sertoli cells may reflect a finely tuned regulatory mechanism in which PAI-1 could limit the excession of the proteolysis.

Localization and distribution of tissue type and urokinase type plasminogen activators and their inhibitors type 1 and 2 in human and rhesus monkey fetal membranes

Fetal membranes consist of 10 distinct layers including components of amnion, chorion and decidua, the latter being of maternal origin. They form mechanically integrated sheets capable of retaining amniotic fluid and play an essential role in protecting fetal growth and development in the pregnant uterus. The extracellular matrix, substrate for plasminogen activators (PAs), is an important supportive framework of the fetal membranes. :Fetal membranes from women with preterm premature rupture of membranes may differ in their protease activity compared with normal membranes. To identify the presence of PAs and their inhibitors (PAI) and their possible role in the process of fetal membrane rupture, this study in investigated the distribution and localization of both protein and mRNA for tissue (t) and urokinase (u) PA and their inhibitors type 1 (PAI-1) and type 2 (PAI-2) in amniochorion of human and rhesus monkey using conventional and. confocal immunofluorescence microscopy. In situ hybridization analysis showed that the distribution and localization of mRNAs for tPA, uPA, PAI-I and PAI-2 were similar in the fetal membranes of human and rhesus monkey; no obvious species difference was observed. Evidence of tPA mRNA was detected in amniotic epithelium, trophoblast cells and nearly all cells of the decidual layer. Strong expression of uPA mRNA was noted in the decidual cells which increased in intensity as the abscission point was approached. Weak staining in chorion laeve trophoblast was also detected. In situ hybridization experiments showed PAI-1 mRNA to be concentrated mainly in the decidual cells, some of which were interposed into the maternal-facing edge of the chorion laeve. Maximal labelling of the decidua occurred towards the zone of abscission. Weak expression of PAI-1 mRNA nas also noted in some cells of the chorion laeve. The distribution of PAI-2 mRNA in amniochorion was also concentrated in the cells of the decidual layer, maximum expression of the mRNA was in the level of abscission. No detectable amount of mRNAs for tPA, uPA, PAI-1 and PAI-2 was found in the fibroblast, reticular and spongy layers. Distribution of the proteins of tPA, uPA and PAI-1 in the fetal membranes of these two species was consistent with the distribution of their mRNA. Anti-PAI-2 immunofluorescence was found to be strongly concentrated in the amniotic epithelium, but PAI-2 mRNA was negative in this layer, suggesting that the epithelium-associated PAI-2 is not of epithelial origin. These findings suggest that a local fibrinolysis in fetal membranes generated by precisely balanced expression of PAs and their inhibitors via paracrine or autocrine mechanisms may play an essential role in fetal membrane development, maturation and in membrane rupture. Following an analysis of the distribution and synthesis of activators and inhibitors it was found that they may play a role in abscission during the third stage of labour. (C) 1998 W. B. Saunders Company Ltd.

An improved microtiter assay for evaluating anti-HIV-I neutralizing antibodies from sera or plasma

Background: The anti-HIV-1 neutralizing antibody assay is widely used in AIDS vaccine research and other experimental and clinical studies. The vital dye staining method applied in the detection of anti-HIV-1 neutralizing antibody has been used in many laboratories. However, the unknown factor(s) in sera or plasma affected cell growth and caused protection when the tested sera or plasma was continuously maintained in cell culture. In addition, the poor solubility of neutral red in medium (such as RPMI-1640) also limited the use of this assay. Methods: In this study, human T cell line C8166 was used as host cells, and 3-(4,5-Dimethyl-2-thiazolyl)- 2,5-diphenyl-2H-tetrazolium bromide (MTT) instead of neutral red was used as vital dye. In order to avoid the effect of the unknown factor( s), the tested sera or plasma was removed by a washout procedure after initial 3 - 6 h culture in the assay. Result: This new assay eliminated the effect of the tested sera or plasma on cell growth, improved the reliability of detection of anti-HIV-1 neutralizing antibody, and showed excellent agreement with the p24 antigen method. Conclusion: The results suggest that the improved assay is relatively simple, highly duplicable, cost-effective, and well reliable for evaluating anti-HIV-1 neutralizing antibodies from sera or plasma.

Hexabromocyclododecane-induced developmental toxicity and apoptosis in zebrafish embryos

Hexabromocyclododecane (HBCD) is widely used as a brominated flame retardant, and has been detected in the aquatic environment, wild animals, and humans. However, details of the environmental health risk of HBCD are not well known. In this study, zebrafish embryos were used to assess the developmental toxicity of the chemical. Four-hour post-fertilization (hpf) zebrafish embryos were exposed to various concentrations of HBCD (0, 0.05, 0.1, 0.5, and 1.0 mg L-1) until 96 h. Exposure to 0.1, 0.5, and 1.0 mg L-1 HBCD significantly increased the malformation rate and reduced survival in the 0.5 and 1.0 mg L-1 HBCD exposure groups. Acridine orange (AO) staining showed that HBCD exposure resulted in cell apoptosis. Reactive oxygen species (ROS) was significantly induced at exposures of 0.1, 0.5, and 1.0 mg L-1 HBCD. To test the apoptotic pathway, several genes related to cell apoptosis, such as p53, Puma, Apaf-1, caspase-9, and caspase-3, were examined using real-time PCR. The expression patterns of these genes were up-regulated to some extent. Two anti-apoptotic genes, Mdm2 (antagonist of p53) and Bcl-2 (inhibitor of Bax), were down-regulated, and the activity of capspase-9 and caspase-3 was significantly increased. The overall results demonstrate that waterborne HBCD is able to produce oxidative stress and induce apoptosis through the involvement of caspases in zebrafish embryos. The results also indicate that zebrafish embryos can serve as a reliable model for the developmental toxicity of HBCD. (C) 2009 Elsevier B.V. All rights reserved.