Construction and Characterization of a cDNA Library from the Pulp of Coconut （Cocos nucifera L.）

To investigate the gene expression profile of endosperm development, a cDNA library was constructed and characterized from the pulp of coconut at different developmental stages. The constructed cDNA library incorporated approximately 1 × 10^7 clones in total, and the size of the insertion fragments ranged from 800 to 2 000 bp. Sequencing results of 100 randomly picked clones showed that the recombination rate was 96%. In subsequent sequence analysis, 41 clones （41%） were homologous to known function proteins, and 23 clones showed high amino acid identity （more than 80%） with the corresponding genes of different plants. Semi-quantitative RT-PCR indicated that oleosin and globulin genes are pulpspecific expression, and have differential expression level in different developmental stage. Clone 29, recognized as homologous to KIAA1239 protein （Homo sapiens）, was observed to occur nine times, indicating that this gene may be over-expressed during the endosperm development stage. However, the homologous protein was found only in mammals, and the detailed function is still unknown. Elucidation of the functional characterization of these genes will be carried out immediately.     

Molecular Characterization and Expression Pattern of Rheb Gene in Inner Mongolia Cashmere Goat （Capra hircus）

As one member of the Ras super family, Rheb is an upstream regulator of mTOR signaling pathway, which regulates the process of cell-growth, proliferation and differentiation. In order to study the relationship between Rheb and mTOR in Inner Mongolian Cashmere goat (Capra hircus) cells, Ras homolog enriched in brain (Rheb) gene cDNA was amplified by RT-PCR. It is 555 bp in length and includes the complete ORF encoding 184 amino acids (GenBank accession no. HM569224). The full cDNA nucleotide sequence has a 99% identity with that of sheep, 98% with cattle and 93% with human while their amino acids sequence shares identity with 98, 97 and 97% of them, correspondingly. The bio informatics analysis showed that Rheb has a Ras family domain, two casein kinase II phosphorylation sites, two ATP/GTP-binding sites motif A (P-loop), a prenyl group binding site (CAAX box). Tissue-specific expression analysis performed by semi-quantitative RT-PCR. The Rheb gene was expressed in all the tested tissues and the highest level of mRNA accumulation was detected in brain, suggesting that Rheb played an important role in goat cells.     

Generation and Analysis of Expressed Sequence Tags （ESTs） from Muscle Full-Length cDNA Library of Wujin Pig

Porcine skeletal muscle genes play a major role in determining muscle growth and meat quality. Construction of a full-length cDNA library is an effective way to understand the expression of functional genes in muscle tissues. In addition, novel genes for further research could be identified in the library. In this study, we constructed a full-length cDNA library from porcine muscle tissue. The estimated average size of the cDNA inserts was 1 076 bp, and the cDNA fullness ratio was 86.2%. A total of 1 058 unique sequences with 342 contigs （32.3%） and 716 singleton （67.7%） expressed sequence tags （EST） were obtained by clustering and assembling. Meanwhile, 826 （78.1%） ESTs were categorized as known genes, and 232 （21.9%） ESTs were categorized as unknown genes. 65 novel porcine genes that exhibit no identity in the TIGR gene index of Sus scrofa and 124 full-length sequences with unknown functions were deposited in the dbEST division of GenBank （accession numbers： EU650784-EU650788, GE843306, GH228978-GH229100）. The abundantly expressed genes in porcine muscle tissue were related to muscle fiber development, energy metabolism and protein synthesis. Gene ontology analysis showed that sequences expressed in porcine muscle tissue contained a high percentage of binding activity, catalytic activity, structural molecule activity and motor activity, which involved mainly in metabolic, cellular and developmental process, distributed mainly in intracellular region. The sequence data generated in this study would provide valuable information for identifying porcine genes expressed in muscle tissue and help to advance the study on the structure and function of genes in pigs.     

Molecular Characterization and Expression Profiles of Myrosinase Gene（RsMyr2） in Radish（Raphanus sativus L.）

Myrosinase is a defense-related enzyme and is capable of hydrolyzing glucosinolates into a variety of compounds, some of which are toxic to pathogens and herbivores. Many studies revealed that a number of important vegetables or oil crops contain the myrosinase-glucosinolate system. However, the related promoter and genomic DNA sequences as well as expression profiles of myrosinase gene remain largely unexplored in radish（Raphanus sativus）. In this study, the 2 798 bp genomic DNA sequence, designated as RsMyr2, was isolated and analyzed in radish. The RsMyr2 consisting of 12 exons and 11 introns reflected the common gene structure of myrosinases. Using the genomic DNA walking approach, the 5′-flanking region upstream of RsMyr2 with length of 1 711 bp was successfully isolated. PLACE and PlantCARE analyses revealed that this upstream region could be the promoter of RsMyr2, which contained several basic cis-regulatory elements including TATA-box, CAAT-box and regulatory motifs responsive to defense and stresses. Furthermore, recombinant pET-RsMyr2 protein separated by SDS-PAGE was identified as myrosinase with mass spectrometry. Real-time PCR analysis showed differential expression profiles of RsMyr2 in leaf, stem and root at different developmental stages（e.g., higher expression in leaf at cotyledon stage and lower in flesh root at mature stage）. Additionally, the RsMyr2 gene exhibited up-regulated expression when treated with abscisic acid（ABA）, methyl jasmonate（MeJA） and hydrogen peroxide（H2O2）, whereas it was down-regulated by wounding（WO） treatment. The findings indicated that the expression of RsMyr2 gene was differentially regulated by these stress treatments. These results could provide new insight into elucidating the molecular characterization and biological function of myrosinase in radish.     

Cloning and Characterization of Genes Coding for Fructan Biosynthesis Enzymes （FBEs）in Triticeae Plants

Fructan is not only a carbon source for storage but also plays an important role as anti-stress agents in many plant species. Complex fructans having both β-（2,1）- and β-（2,6）-linked fructosyl units accumulate in Triticeae plants commonly. Three enzymes （sucrose： sucrose 1-fructosyltransferase, 1-SST, EC： 2.4.1.99; sucrose： fructan 6-fructosyltransferase, 6- SFT, EC： 2.4.1.10; and fructan： fructan 1-fructosyltransferase, 1-FFT, EC： 2.4.1.100） were involved in fructan biosynthesis in Triticeae plant species. We successfully isolated these genes from tetraploid wheat （Triticum turgidum, genotype： AABB）, common wheat （Triticum aestivum L., genotype： AABBDD） and three wild relatives of common wheat, Triticum urartu Thum. （the origin of the AA genome）, Aegilops speltoides （Tausch） Gren. （the putative source of the SS genome） and Aegilops tauschii Coss. （the source of the DD genome）. Sequence analysis revealed that all the FBEs （fructan biosynthetic enzymes） had three highly conserved functional motifs except 1-SST （EU981912） from tetraploid wheat species only with conserved DPNG. Low pI （isoelectric point） and potential N-glycosylation sites were predicted, which were crucial for protein compartmentation and post-translational process. Analysis on subcelluar localization signals showed that only 6-SFT had vacuolar-directed signal. Sequences alignment result showed that 1-SST and 1-FFT were more conservative and had closer relationship each other, while 6-SFT was more active during the evolution processing. According to the syntenic relationship between wheat and rice genome, FBEs were predicated to be located on the homeologous group 6 and group 2 chromosomes. Expression profile confirmed that expression of all the three FBEs were drought-stress induced. This study can assist to establish a useful theoretical platform for cold- or drought-tolerant improvement of wheat by modulating FBEs expression.     

Cloning and Characterization of a Somatic Embryogenesis Receptor-Like Kinase Gene in Cotton （Gossypium hirsutum）

A novel gene,GhSERK1,was identified in cotton.It encoded a protein belonging to the somatic embryogenesis receptorlike kinase (SERK) family.The genomic sequence of GhSERKI was 6920 bp in length,contain...     

草鱼受精卵菌群的研究

对草鱼受精卵及所处水体的好氧及兼性厌氧菌的数量和组成进行了研究.结果表明:草鱼受精卵菌群的数量随受精卵的发育呈递增趋势,刚受精时每个受精卵的细菌数量为1.24×101cfu,出膜前增加到1.41×105cfu.水体中细菌的数量相对稳定,为1.05×104～3.85×104cfu/mL,只在受精卵出膜前的阶段急剧增加,为1.32×106cfu/mL.随受精卵的发育,受精卵的优势菌群先为气单胞菌属(Aeromonas)、肠杆菌科(Enterobac-teriaceae)、微球菌属(Micrococcus)和棒杆菌属(Corynebacterium),后为莫拉氏菌属(Moraxella)和不动杆菌属(Acinetobacter).水体中的优势菌群为气单胞菌属(Aeromonas)、肠杆菌科(Enterobacteriaceae)和莫拉氏菌属(Moraxella).受精卵的优势菌群与其所处水体的优势菌群基本相同.     

天然复合脱腥保鲜液对草鱼的脱腥保鲜效果

研究比较了黄酒、姜葱蒜以及姜葱蒜-黄酒复合脱腥保鲜液对于新鲜草鱼鱼肉的脱腥效果,并选用脱腥效果最好的姜葱蒜-黄酒复合脱腥保鲜液应用于新鲜草鱼鱼肉的保鲜.通过测定货架期草鱼鱼肉的pH值、挥发性盐基总氮、TBA值、菌落总数以及感官评定,研究了脱腥保鲜液对于草鱼鱼肉货架期品质的影响.结果表明,该复合脱腥保鲜液能够有效地保持鱼肉的货架期品质,延缓鱼肉pH值、TVBN值、TBA值以及菌落总数的升高.     

草鱼的体液免疫应答及抗体产生细胞

以草鱼出血病病毒和牛血清白蛋白（ＢＳＡ）为抗原妆种草鱼,比较了草鱼对两种抗原的体液免疫应答情况。结果显示,在接种了ＢＳＡ的草鱼中,１４ｄ后检测至抗体,其效价在２８ｄ达到峰值;而接种了出血病病毒的草鱼,在２１ｄ后检测到抗体,抗体效价在３５ｄ达到峰值。用ＦＴＴＣ－ＢＳＡ检测免疫ＢＳＡ草鱼的抗体产生细胞,探讨了抗体产生效价与抗体细胞数量的关系。     

玉米淀粉及氯化钙对草鱼鱼糜热凝胶特性的影响

将玉米淀粉及氯化钙添加到草鱼鱼糜中,通过对鱼糜热凝胶pH值、色泽、质构特性以及持水性的测定,评价了玉米淀粉及氯化钙对于鱼糜热凝胶特性的影响。结果表明,氯化钙能使鱼糜热凝胶白色度升高,弹性及凝聚性增强而降低其pH值及持水性。玉米淀粉成分的存在能够增强鱼糜凝胶的持水性。添加玉米淀粉与2g/kg氯化钙的组合能够一定程度上改善鱼糜热凝胶的色泽、质构特性以及持水性。     

草鱼脂多糖结合蛋白的分离纯化(英文)

脂多糖结合蛋白(lipopolysaccharide binding proteins ,LBP)是机体对革兰氏阴性菌感染产生的可溶性急性期蛋白,结合并提呈LPS给细胞表面的模式识别受体,激发免疫反应。在本试验中,用低浓度嗜水气单胞菌( Aeromonas hydrophila)感染草鱼(Ctenopharyngodon idellus)24 h后,抽取全血,离心,获得血浆。结合LBP活性检测,经硫酸铵沉淀、CMSephadex C-50阳离子交换层析和DEAE Sephadex A-25阴离子交换层析后,分离纯化得到LBP。该蛋白对异硫氰酸荧光素标记的脂多糖(Fluorescein isothiocyanate labeled li-popolysaccharide ,FITC-LPS)有较强的结合能力。在SDS-PAGE电泳后,考马斯亮蓝染色可见3条明显的带,分子量大约为68、53和48 kDa。同时探索一条简便分离纯化脂多糖结合蛋白的方法,为进一步研究LBP的功能奠定了基础。     

"肝胆综合症"草鱼血液生化特性及组织结构变化

采用组织学和生物化学的方法,对肝脏肿大、变色草鱼的血液生化指标和肝脏组织结构进行研究。与肝脏正常草鱼相比,肝脏变异组草鱼肝胰脏颜色变淡、甚至变绿;肝细胞发生一定程度的脂肪变性或水样变性,甚至胀亡坏死;血清中天冬氨酸氨基转移酶（AST）、丙氨酸氨基转移酶（ALT）、直接胆红素（DB）、总胆红素（TB）、甘油三酯（TG）、肌酐（CRE）均显著升高（P〈0.05）,血清尿素氮（BUN）和胆固醇（CHO）含量有增加趋势但不显著（P〉0.05）,血糖（UGLU）含量和碱性磷酸酶（ALP）的含量无显著性差异（P〉0.05）;超氧化物歧化酶（SOD）显著性降低（P〈0.05）,补体C3、C4的活性值都有不同程度的降低（P〉0.05）;肝糖原显著性降低（P〈0.05）,肝中脂肪含量有所增加但不显著（P〉0.05）。结果表明,肝脏外观产生变异草鱼的肝脏有不同程度的损伤,肾脏也受到一定的损害,同时免疫系统也受到破坏。     

高效氯氰菊酯对草鱼血清和脾脏酸性磷酸酶活性的影响

研究了暴露在不同浓度高效氯氰菊酯下草鱼(Ctenopharyngodon idellas)血清和脾脏中酸性磷酸酶(ACP)的活性变化.将高效氯氰菊酯浓度设5组,包括1个对照组(0μg/L)和4个处理组(0.5、1.0、3.0、5.0μg/L),每组分别于暴露1、5、12d时取样,测定血清和脾脏ACP的活性.结果显示,暴露1d时,血清ACP活性各处理组显著下降;脾脏ACP活性0.5μg/L组和1.0μg/L组显著升高,3.0μg/L组和5.0μg/L组显著下降.暴露5d时,血清ACP活性1.0μg/L组显著上升,3.0μg/L组和5.0μg/L组显著下降;脾脏ACP活性1.0、3.0和5.0μg/L组显著下降.暴露12d时,血清ACP活性1.0、3.0和5.0μg/L组显著下降;脾脏ACP活性各处理组均显著下降.ACP活性的变化反映了高效氯氰菊酯能够影响鱼体的代谢平衡,对鱼体具有毒害作用;同时,血清和脾脏的ACP活性对高效氯氰菊酯早期污染的敏感性可以作为监测水体中此类化学品污染的一个特异性标志物.     

草鱼母源免疫的初步研究

用酚灭活的柱状嗜纤维菌对草鱼雌亲鱼每月注射１次，连续注射７次。通过检测草鱼雌亲鱼血液中吞噬细胞的吞噬活性、血清中凝集抗体效价、受精卵和鱼苗中凝集抗体效价，首先证明了草鱼雌亲鱼对免疫原能产生较强的免疫应答，其凝集抗体可以通过受精卵而传递给仔代，而且这种凝集抗体能在孵化后的鱼苗体内持续存在２７ｄ以上。然而，用活菌对１７４日龄的鱼种攻毒的试验结果表明，免疫组与对照不存在显著差异。     

微囊藻毒素-LR对草鱼肝脏超微结构的亚急性毒性影响

微囊藻毒素-LR(microcystin-LR, MC-LR)是一种由蓝藻水华产生的环状肝毒素,对鱼类存在不可忽视的影响.草鱼(Ctenopharyngodon idella)经腹腔注射MC-LR(纯度>98%,50 μg/kg体重),在注射后1、2、7、14和21 d采集肝脏样品,用透射电镜的方法研究发现,MC-LR除可导致线粒体水肿、内质网扩张外,还可引起草鱼肝脏细胞连接间隙增宽,胆汁淤积,炎性细胞浸润,细胞质中大量脂滴及脂褐素、溶酶体增多等一系列的病变;但在注射MC-LR 21 d后草鱼肝脏细胞连接又基本恢复正常.结果表明,MC-LR在低剂量下除了对膜系结构存在影响外,对细胞连接间隙等还存在影响,然而随着时间的延长,这些病变可逐渐修复.     

普通草鱼与脆肉鲩肝胰脏游离氨基酸的差异

为了探明脆肉鲩(脆化饲养法)肉质脆化的机理,对普通草鱼(常规饲养法)和脆肉鲩的肝胰脏进行预处理,用毛细管气相色谱法对其游离氨基酸的组成和含量进行测定．结果表明：普通草鱼和脆肉鲩的肝胰脏游离氨基酸组成相同,但含量差异显著,100g鲜样中游离氨基酸总含量分别为194．11mg和373．23mg,游离必需氨基酸总含量分别为82．62mg和191．55mg．这种变化说明在草鱼的饲养过程中,改变其食物组成导致了草鱼肝胰脏游离氨基酸含量的改变。