运用减法杂交、差异筛选与信使RNA差别显示聚合酶链式反应克隆冬小麦春化相关基因

对于某些一年生或二年生高等植物，春化作用是诱导其成花的一个重要的环境因子。冬小麦春化进程中存在着一个核酸代谢的关键期，利用分子生物学技术分离特异表达的基因是研究春化诱导成花机理的一个突破口。 利用TRIzol试剂快速提取冬小麦燕大1817(Triticum aestivum L. cv Yanda 1817)未春化、春化4d、春化20d、5d脱春化的胚芽中的总RNA，去除污染的DNA后，将引物P_1(5'TTTTTTTTTTTCA3')、P_2(5'TTTTTTTTTTCC3')与10个碱基的随机引物OPF_1-OPF_(20)、OPG_1-OPG_(20)组成80个引物对，对不同来源的RNA进行差别显示，共显示了大约10,000种mRNA，结果发现了两个仅在春化20d这一关键期表达而在未春化、春化4d、5d脱春化时不表达的春化相关基因(VRG)VRG49与VRG54。Northern分析进一步表明这两个基因仅与春化20d的冬小麦RNA有杂交信号。将VRG49与VRG54亚克隆于pGEM-4Z载体上，利用T_7测序系统获得了VRG49和VRG54的DNA序列，它们的长度分别为307bp与169bp。 春化21d的冬小麦京冬1号(T. aestivum L. cv Jingdong No. 1)胚芽的mRNA在逆转酶作用下反转录成sscDNA杂交，将过量的未春化、脱春化的mRNA与sscDNA杂交，运用磁珠法分离出未杂交上的sscDNA，以特异的sscDNA为模板，用DNA聚合酷I合成了dscDNA。通过对dscDNA内部EcoRI位点的甲基化、末端补平、EcoRI接头的安装、连接进入λgt10载体的EcoRI位置，以及运用包装系统进行体外包装，建立了库容为4 * 10~6pfu的富集低温诱导的冬小麦cDNA噬菌体文库。用来源于未春化、春化21d、脱春化的冬小麦mRNA合成3种cDNA探针，对噬菌斑进行原位杂交，结果筛选出了3个春化相关基因(VRG)VRG79、VRG111和VRG231。Dot blotting与Northern分析表明VRG79仅在冬小麦春化关键期21d表达。运用PCR方法从λgt10DNA中扩增出VRG79片断并亚克隆于PUC18载体上，通过T_7测序系统获得了VGR79的序列，其包括349个碱基。 通过Internet将VRG49、VRG54、VRG79与GenBank、EMBL、DDBJ、PBD中的序列进行同源性分析，结果发现这些基因至少是在植物中新发现的基因，对这些基因推测的一些功能也进行了讨论。

小麦抗禾谷孢囊线虫（ Heterodera avenae）相关基因的研究

禾谷孢囊线虫严重影响禾谷类作物的产量，在小麦中由禾谷孢囊线虫引起的产量损失可达30-100%。尤其在澳大利亚、欧洲、印度和中东危害严重，目前禾谷孢囊线虫已成为危害我国作物的主要病源。控制禾谷孢囊线虫的方法主要有：作物轮作、杀线虫剂、寄主抗性等等，其中基因工程方法培育抗线虫小麦品种被认为是最经济有效的方法。分离抗禾谷类孢囊线虫基因对揭示抗性基因结构与功能及其表达调控具有重要意义。 尽管小麦是重要的粮食作物，在小麦中已发现的抗禾谷孢囊线虫的基因很少，而比其近缘属如节节麦、易变山羊草、偏凸山羊草中含有丰富的抗源。目前已鉴定出禾谷孢囊线虫抗性位点Cre，并发现了9个禾谷孢囊线虫抗性基因(Cre1,2, 3, 4, 5, 6, 7, 8, and R) ，其中只有Cre1和Cre8直接从普通小麦中获得。从节节麦中获得的Cre3基因能最有效的控制线虫数量，其次是Cre1和Cre8。这些基因的克隆对于了解禾谷孢囊线虫抗性机制及进一步的育种应用都是非常关键的。然而，目前为止仅有Cre3基因通过图位克隆的方法从节节麦中被分离得到。该基因已被克隆得到的多数线虫抗性基因一样均属于核苷酸结合位点区（NBS）－亮氨酸重复序列区（LRR）基因家族。目前，已有很多抗性基因被分离，这些已知的NBS-LRR类抗性基因的保守序列为应用PCR的方法克隆新的抗性基因提供了可能。 因此本课题的目的是采用保守区同源克隆、3′RACE 和5′RACE 等方法从抗禾谷孢囊线虫小麦-易变山羊草小片段易位系E10 中克隆小麦抗禾谷孢囊线虫基因全序列，进而通过半定量PCR 和荧光定量PCR 研究该基因的表达模式。同时通过mRNA 差别显示技术和任意引物PCR（RAP-PCR）技术分离克隆植物禾谷孢囊线虫抗性基因及其相关基因，为阐明植物抗病性分子机制以及改良作物抗病性和作物育种提供基础，为通过分子标记辅助育种和基因工程方法实现高效、定向转移抗病基因到优良小麦品种奠定了重要的理论和物质基础。主要研究结果： 1． 本实验根据此前从抗禾谷孢囊线虫材料E-10 扩增得到的与来自节节麦的抗禾谷孢囊线虫Cre3 基因及其他的NBS-LRR 类抗性基因的NBS 和LRR 保守区序列设计了两对特异性引物，从E10 中扩增到532bp 和1175bp 的两个目标条带，它们有一个32bp 的共同序列，连接构成总长为1675bp 的NBS-LRR 编码区（命名为RCCN）。根据RCCN设计引物，利用NBS-LRR区序列设计引物，通过5′RACE 和3′RACE 技术采用3′-Full RACE Core Set(TaKaRa)和5'-Full RACE Kit (TaKaRa)试剂盒，反转录后通过嵌套引物GSP1 和GSP2 分别进行两轮基因特异性扩增，分别将NBS_LRR 区向5′端和3′端延伸了1173bp 和449bp，并包含了起始密码子和终止密码子。根据拼接的得到的序列重新设计引物扩增进行全基因扩增的结果与上面获得的一致。拼接后得到全长2775 bp 的基因序列（记作CreZ, GenBank 号：EU327996）。CreZ 基因包括完整的开放阅读框，全长2775 bp，编码924个氨基酸。序列分析表明它与已知的禾谷孢囊线虫抗性基因Cre3的一致性很高，并且它与已经报到的NBS-LRR 类疾病抗性基因有着相同的保守结构域。推测CreZ基因可能是一个新的NBS-LRR 类禾谷孢囊线虫抗性基因，该基因的获得为通过基因工程途径培育抗禾谷孢囊线虫小麦新品种奠定了基础，并为抗禾谷孢囊线虫基因的调控表达研究提供了参考。 2． 通过半定量PCR和SYBR Green荧光定量PCR技术对CreZ基因的相对表达模式进行了研究。以α-tubulin 2作为参照，采用半定量PCR 分析CreZ 基因在不同接种时期1d, 5d, 10, 15d 的E-10的根和叶的的表达情况。在内参扩增一致的条件下，CreZ 在E-10的根部随着侵染时间的增加表达量有明显的增加，在没有侵染的E-10的根部其表达量没有明显变化，而在叶中没有检测表达，说明该基因只在抗性材料的根部表达。SYBR Green定量PCR分析接种前后E10根部基因CreZ基因的表达水平为检测CreZ基因的表达建立了一套灵敏、可靠的SYBRGreen I 荧光定量PCR 检测方法。接种禾谷孢囊线虫后E10根内CreZ基因的相对表达水平显著高于接种前。随接种时间的延长持续增加，最终CreZ基因的相对表达量达到未接种的对照植株的10.95倍。小麦禾谷孢囊线虫抗性基因CreZ的表达量与胁迫呈正相关，表明其与小麦的的禾谷孢囊线虫抗性密切相关，推测CreZ基因可能是一个新的禾谷孢囊线虫候选抗性基因。 3． 针对小麦基因组庞大、重复序列较多，禾谷孢囊线虫抗性基因及其相关基因的片断难以有效克隆的问题，通过mRNA 差别显示技术及RAP-PCR 技术分离克隆植物禾谷孢囊线虫抗性及其相关基因。试验最终得到154 条差异表达条带，将回收得到的差异条带的二次PCR 扩增产物经纯化后点到带正电的尼龙膜上，进行反向Northern 杂交筛选，最终筛选得到102 个阳性差异点。将其中81 个进行测序，并将序列提交到Genbank 中的dbEST 数据库，分别获得登录号(FE192210 -FE192265，FE193048- FE193074 )。序列比对分析发现，其中26 个序列与已知功能的基因序列同源；有28 条EST 序列在已有核酸数据库中未找到同源已知基因和EST，属新的ESTs 序列；另外27 个EST 序列与已知核酸数据库中的ESTs 具有一定相似性，但功能未知。其所得ESTs 序列补充了Genbank ESTs 数据库，为今后进一步开展抗禾谷类孢囊线虫基因研究工作打下了基础。结合本试验功能基因的相关信息，对小麦接种禾谷孢囊线虫后产生的抗性机制进行了探讨。接种禾谷孢囊线虫后植物在mRNA 水平上的应答是相当复杂的，同时植物的抗病机制是一个复杂的过程，涉及到多个代谢途径的相互作用。 The cereal cyst nematode (CCN), Heterodera avenae Woll, causes severe yieldreductions in cereal crops. The losses caused by CCN can be up to 30-100% in somewheat fields. At present, cereal cyst nematode has become the major disease sourcein China and it also damaged heavily in Australia, Europe, India and Middle East.The damage caused by CCN can be mitigated through several methods, includingcrop rotation, nematicide application, cultural practice, host resistance, and others.Of these methods, incorporating resistance genes into wheat cultivars and breedingresistant lines is considered to be the most cost-effective control measure forreducing nematode populations. Although wheat is an economically important crop around the world, far fewergenes resistant to CCN were found in wheat than were detected in its relatives, suchas Aegilops taucchi, Aegilops variabilis and Aegilops ventricosa. Cloning these genesis essential for understanding the mechanism of this resistance and for furtherapplication in breeding. Because of the huge genome and high repeat sequencescontent, the efficient methods to clone genes from cereal crops, are still lacking. A resistance locus, Cre, has been identified and 9 genes resistant to CCN (designatedCre1, 2, 3, 4, 5, 6, 7, 8, and R) have been described, in which Cre1 and Cre8 werederived directly from common wheat. The Cre3 locus, which was derived from Ae.tauschii, has the greatest impact on reducing the number of female cysts, followed byCre1 and Cre8. Cloning these genes is essential for understanding the mechanism ofthis resistance and for further application in breeding. However, to this point, only Cre3, a NBS-LRR disease resistance gene, has been obtained through mappingcloning in Ae. tauschii. The majority of nematode resistance genes cloned so far belong to a super familywhich contains highly conserved nucleotide-binding sites (NBS) and leucine-richrepeat (LRR) domains. To date, many NBS-LRR resistance genes have been isolated.The conserved sequences of these recognized NBS-LRR resistance genes provide thepossibility to isolate novel resistance genes using a PCR-based strategy. The aim of the present study was to clone the resistance gene of CCN fromWheat/Aegilops variabilis small fragment chromosome translocation line E10 whichis resistant to CCN and investigate the espression profiles of this gene withsemi-quantitative PCR and real-time PCR. Another purpose of this study is cloningthe relational resistance gene for CCN by mRNA differential display PCR andRAP-PCR. These works will offer a foundation for disease defence of crop andbreeding and directional transferring resistance gene into wheat with geneengineering. Primary results as following: 1．According to the conversed motif of NBS and LRR region of cereal cystnematode resistance gene Cre3 from wild wheat (Triticum tauschlii) and the knownNBS-LRR group resistance genes, we designed two pairs of specific primers for NBSand LRR region respectively. One band of approximately 530bp was amplified usingthe specific primers for conversed NBS region and one band of approximately 1175bpwas amplified with the specific primers for conversed LRR region. After sequencing,we found that these two sequences included 32bp common nucleotide having 1675bpin total, which was registered as RCCN in the Genbank. Based on the conservedregions of known resistance genes, a NBS-LRR type CCN resistance gene analog wasisolated from the CCN resistant line E-10 of the wheat near isogenic lines (NILs), by5′RACE and 3′ RACE.designated as CreZ (GenBank accession number: EU327996) .It contained a comlete ORF of 2775 bp and encoded 924 amino acids. Sequencecomparison indicated that it shared 92% nucleotide and 87% amino acid identitieswith those of the known CCN-resistance gene Cre3 and it had the same characteristic of the conserved motifs as other established NBS-LRR disease resistance genes. 2． Usingα-tubulin 2 as exoteric reference, semi-quantitative PCR and real-timePCR analysis were conducted. The expression profiling of CreZ indicated that it wasspecifically expressed in the roots of resistant plants and its relative expression levelincreased sharply when the plants were inoculated with cereal cyst nematodes. therelative expression level of the 15days-infected E10 is the 10.95 times as that ofuninfected E10,ultimately. It was inferred that the CreZ gene be a novel potentialresistance gene to CCN. 3．We cloned the relational resistance gene for CCN by mRNA differentialdisplay PCR and arbitrarily primed PCR fingerprinting of RNA from wheat whichpossess huge and high repeat sequence content genomes. Total 154 differentialexpression bands were separated and second amplified by PCR. The products werenylon membrane. The 102 positive clones were filtrated by reverse northern dot blotand 81 of those were sent to sequence. The EST sequences were submitted toGenbank (Genbank accession: FE192210 - FE192265, FE193048 - FE193074). Thesequences alignment analysis indicated 26 of them were identical with known genes;28 were not found identical sequence in nucleic acid database; another 27 ests wereidentical with some known ests, but their functions were not clear. These ESTsenriched Genbank ESTs database and offered foundation for further research ofresistance gene of CCN.

Differential gene expression of protein kinases in oocytes between natural gynogenetic silver crucian carp and amphimictic crucian carp

A modified mRNA differential display method has been applied to studying differential expression of protein kinase genes in oocytes between natural gynogenetic silver crucian carp and amphimictic crucian carp. Total RNA was reverse transcribed using downstream 3' primers T(12)MA, T(12)MG and T12MC respectively. Then the reverse transcription products were amplified using upstream 5' kinase-specific primer designed according to protein kinase conserved sequence. The PCR products had different patterns and numbers of: cDNA bands on polyacrylamide:gel. Totally 21 cDNAs fragments were recovered and cloned. Two of them were confirmed to be particularly expressed in oocytes of amphimictic crucian carp, and another was specific for gynogenetic silver crucian carp.

羊草细胞差异表达基因分析与遗传转化方法研究

羊草（Leymus chinensis (Trin.) Tzvel），隶属禾本科赖草属，是欧亚大陆草原区东部重要建群种之一。羊草是牧草之王，是我国比较有优势的战略性生物资源，对我国北方畜牧业的发展以及生态环境的保育均具有重要意义。近年来，由于缺乏科学管理、过度放牧等不利影响，加之羊草本身固有的“三低”问题（即抽穗率低、结实率低、发芽率低）已对羊草生物多样性维持构成了严重的威胁，限制了我国人工草地建设和天然草地的改良及沙化治理的步伐。因此，如何通过细胞、分子生物学以及生物技术手段改良羊草、快速评价和创造新的种质;如何加快育种进程便成为当前亟待解决的问题。本文围绕这些问题开展了系统的研究并取得如下结果： 1. 建立了羊草离体培养再生体系，并研究了影响愈伤组织诱导和植株再生的因素，影响植株再生的主要因素为激素配比和基因型。将3～5mm的幼穗接种到含有2,4-D 0～5.0mg/L的N6基本培养基上，随着2,4-D浓度的变化，愈伤组织诱导率不同，最高诱导率为93.21%（基因型C6）、最低为33.35%（吉生1号）;愈伤组织在N6(大)＋B5(微)＋KT1.0mg/L+BA1.0mg/L的培养基上可以分化出芽，并在1/2MS培养基上生根。羊草基因型W4不同幼穗诱导的愈伤组织在继代培养过程中其生长、褐化死亡等方面存在着差异;在分化培养过程中，不同幼穗的愈伤组织最高分化率为9.24%，最低分化率为5.26%。 2. 对来自同一基因型不同幼穗的愈伤组织中差异表达的基因进行了研究。采用DDRT-PCR技术对其差异表达的基因进行了分离，通过银染技术显示差异片段。将得到的差异片段进行回收、克隆测序，得到两个差异片段序列，经过序列分析表明，其中一个片段是与水稻翻译延伸因子eEF-1基因高度同源;另一差异片段与水稻谷胱甘肽转移酶GST基因高度同源。 3. 建立了羊草遗传转化方法。在获得羊草离体培养再生体系的基础上，采用基因枪法对羊草两个基因型进行转抗除草剂基因（PAT）的研究。对分别来自基因型W4和C3的愈伤组织各1430和1850块进行转化。在附加1.0mg/L PPT的培养基上进行一系列的筛选培养，共获得了23株再生苗，经过生根筛选培养，得到5株抗性苗，3株来自基因型W4，2株来自基因型C3。对5株植株进行PCR和Southern 检测，得到2株阳性苗，均来自基因型W4，对阳性植株经过无性繁殖得到的无性系进行PCR检测及Basta耐受性鉴定，外源基因可以在其无性系稳定遗传并表达，无性系除对Basta具有抗性外，其表型特征与对照无明显区别。

Residual plastids of bleached mutants of Euglena gracilis and their effects on the expression of nucleus-encoded genes

Bleached mutants of Euglena gracilis were obtained by treatment with ofloxacin (Ofl) and streptomycin (Sm) respectively. As shown by electron microscopy, the residual plastids contain prothylakoids in an Ofl mutant, and the highly developed and tightly stacked membranous structure found in cells of two Sm, mutants. Nine genes of the plastid genome were examined with PCR, showing that ribosomal protein genes and most other plastid genes were lost in all but one Sm mutant. Using differential display and RT-PCR, it was shown that chloroplast degeneration could cause changes in transcription of certain nucleus-encoded genes during heterotrophic growth in darkness.

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。

牛耳草光合作用的脱水保护和复苏机理

本文以复苏植物牛耳草成熟植株的离体叶片为实验材料，以光合作用、蔗糖、抗氧化剂系统和离子渗漏等在脱水复苏过程中的变化为切入点，从生理生化水平上探讨其耐脱水复苏的机制;同时应用mRNA差异显示技术，从分子水平上探讨其耐脱水复苏的机制。 牛耳草叶片光系统II光化学活性参数和叶黄素循环色素在脱水复苏过程中的变化结果表明，极微弱光强（3μmol.m-2.s-1）下，脱水8天的牛耳草叶片诱导了叶黄素循环，叶黄素循环可能介导了牛耳草叶片脱水过程中的光保护作用。 利用不同浓度的磷酸盐溶液处理牛耳草叶片的结果表明，0.1mol/L以上的磷酸盐溶液对牛耳草叶片具有损伤作用，极大的影响了其光系统II的光化学活性，使得牛耳草叶片在脱水后不能很好的复苏。 牛耳草叶片在脱水复苏过程中，抗坏血酸（AsA）、还原型谷胱甘肽（GSH）和蔗糖含量在脱水时很快增加，复苏时又迅速恢复到原来水平，表明它们可能对脱水的牛耳草叶片具有保护作用，但对复苏的牛耳草叶片可能不重要;其离子渗漏情况表明质膜结构的完整性和稳定性在脱水复苏过程中能得到很好的保持，这可能是其耐脱水复苏的重要机制之一。 利用mRNA差异显示技术分离到牛耳草叶片脱水过程中一些脱水和磷酸盐特异诱导表达的cDNA。对其中5个脱水特异诱导表达和3个磷酸盐特异诱导表达的cDNA进行克隆测序、同源性探测和Northern 杂交检测表明，牛耳草脱水过程中诱导表达的基因可能涉及到脱水胁迫的信号转导、调节基因的级联和结构基因产物调节细胞结构在脱水胁迫中的稳定性等。

Regulation of zygotic gene activation in the preimplantation mouse embryo: Global activation and repression of gene expression

Superimposed on the activation of the embryonic genome in the preimplantation mouse embryo is the formation of a transcriptionally repressive state during the two-cell stage. This repression appears mediated at the level of chromatin structure, because it is reversed by inducing histone hyperacetylation or inhibiting the second round of DNA replication. We report that of more than 200 amplicons analyzed by mRNA differential display, about 45% of them are repressed between the two-cell and four-cell stages. This repression is scored as either a decrease in amplicon expression that occurs between the two-cell and four-cell stages or on the ability of either trichostatin A tan inhibitor of histone deacetylases) or aphidicolin tan inhibitor of replicative DNA polymerases) to increase the level of amplicon expression. Results of this study also indicate that about 16% of the amplicons analyzed likely are novel genes whose sequence doesn't correspond to sequences in the current databases, whereas about 20% of the sequences expressed during this transition likely are repetitive sequences. Lastly, inducing histone hyperacetylation in the two-cell embryos inhibits cleavage to the four-cell stage. These results suggest that genome activation is global and relatively promiscuous and that a function of the transcriptionally repressive state is to dictate the appropriate profile of gene expression that is compatible with further development.

Migration of mouse antibody-secreting hybridoma cells from blood to genital tract and its regulation by sex hormones are associated with the differential expression patterns of adhesion molecules and chemokines in the tract rather than in the antibody-secreting cells

To understand better the molecular mechanisms of differential migration of antibody-secreting cells (ASCs) into mouse genital tracts, and regulation by sex hormones, surface markers, hormone receptors and adhesion molecules in mouse SG2 and PA4 hybridoma cells, respectively, secreting IgG2b and polymeric IgA antibody were detected by flow cytometry or RT-PCR. Semiquantitative RT-PCR was also used for measuring mRNA expression of adhesion molecules and chemokines (VCAM-1, ICAM-1, P-selectin, JAM-1 and CXCL12) in genital tracts of various adult mouse groups. The mRNAs of androgen receptor, estrogen receptor beta and CXCR4 were expressed in the ASCs. Sex hormones had no effect on expression of these molecules in ASCs. Except for VCAM-1, mRNA of all examined genes was expressed in normal mouse genital tracts. The mean of relative amounts of ICAM-1 and CXCL12 mRNA in all examined organs of females were higher (2.1- and 1.9-fold) than those in males. After orchiectomy or ovariectomy, the expression of ICAM-1, CXCL12 and P-selectin mRNA in the examined organs increased, except JAM-1 in male and CXCL12 in female. Sex hormone treatment recovered the changes to normal levels of mRNA expression in many examined genital tissues. In combination with our previous work, preferential migration of ASCs into female genital tract and regulation of migration by sex hormones are associated with expression patterns of adhesion molecules and chemokines in genital tract rather than in ASCs. (C) 2006 Elsevier Ireland Ltd. All rights reserved.

Identification of differential transcript profiles between mutual crossbred embryos of zebrafish (Danio rerio) and Chinese rare minnow (Gobiocypris rarus) by cDNA-AFLP

The crosstalk between naive nucleus and maternal factors deposited in egg cytoplasm before zygotic genome activation is crucial for early development. In this study, we utilized two laboratory fishes, zebrafish (Danio rerio) and Chinese rare minnow and Chinese rare minnow (Gobiocypris rarus), to obtain mutual crossbred embroys and examine the interaction between nucleus and egg cytoplasm from different species. Although these two types of crossbred embryos originated from common nuclei, various developmental capacities were gained due to different origins of the egg cytoplasm. Using cDNA amplified fragment length polymorphism (cDNA-AFLP), We Compared transcript profiles between the mutual crossbred embryos at two developmental stages (50%- and 90%-epiholy). Three thousand cDNA fragments were generated in four cDNA pools with 64 primer combinations. All differently displayed transcript-derived fragments (TDFs) were screened by (lot blot hybridization, and the selected sequences were further analyzed by semi-quantitative RT-PCR and quantitative real-time RT-PCR. Compared with ZR embryos, 12 genes were up-regulated and 12 were down-regulated in RZ embryos. The gene fragments were sequenced and subjected to BLASTN analysis. The sequences encoded various proteins which functioned at various levels of proliferation, growth, and development. One gene (ZR6), dramatically down-regulated in RZ embryos, was chosen for loss-of-function study; the knockdown of ZR6 gave rise to the phenotype resembling that of RZ embryos. (c) 2008 Elsevier Inc. All rights reserved.

Isolation and identification of a novel WSSV nucleocapsid protein by cDNA phage display using an scFv antibody

In a previous study, a scFv phage display library against white spot syndrome virus (WSSV) was constructed and yielded a clone designated A I with conformational specificity against native but not denatured viral antigen. Although the clone A1 has been used successfully as a diagnostic antibody, its precise target antigen has not been elucidated. A different strategy was adopted involving the construction of a second T7 phage display library utilizing mRNA isolated from shrimp infected with WSSV. Following RT-PCR and T7 phage library construction, phages displaying the candidate epitope were selected with A I scFv. Since successive enrichment steps were not associated with an increased titer of the phages, enrichment after successive tests was confirmed by PCR resulting in the prefer-red selection of a specific DNA sequence encoding a novel nucleocapsid protein WSSV388. Immune electron microscopy revealed that WSSV388 is located on the nucleocapsid. This result demonstrated that unknown antigen could be identified by phage display using the epitope conformation dependent scFv. (c) 2006 Elsevier B.V. All rights reserved.

Differential expression of thyroid-stimulating hormone beta subunit in gonads during sex reversal of orange-spotted and red-spotted groupers

We have cloned and characterized the full-length cDNA encoding thyroid-stimulating hormone beta-subunit (TSHbeta) from orange-spotted grouper Epinephelus coioides. It contains 913 nucleotides with an open reading frame encoding 146 amino acids with a 20 amino acid signal peptide. The grouper mature TSHbeta has 75, 70, 61, 59, 41, 42 and 40% identities to that of rainbow trout, Atlantic salmon, zebrafish, European eel, chicken. mouse and human, respectively. RT-PCR analysis indicated that the TSHbeta mRNA was expressed abundantly not only in pituitary but also in gonads. A more interesting finding is to reveal the differential TSHbeta expressions between the ovaries and the transitional gonads or testes in natural individuals of orange-spotted grouper and red-spotted grouper Epinephelus akaara, and in artificial sex reversal individuals of red-spotted grouper induced by MT feeding. In situ hybridization localization provided direct evidence that the TSHbeta was transcribed in the germ cells. In the growing oocytes, the TSHbeta transcripts were concentrated on the ooplasm periphery. In testicular tissues, the intensively expressed TSHbeta cells were found to be spermatogonia and spermatocytes in the spermatogenic cysts. This is the first report of a TSHbeta expressed in the gonads of any vertebrates in addition to the expected expression in the pituitary, and it expresses more transcripts in the gonads during sex reversal or testis than in the ovaries both in E. coioides and E. akaara. Importantly, the TSHbeta identification in germ cells allows us to further investigate the functional roles and the molecular mechanisms in gametogenesis of groupers, especially in sex reversal and in spermatogenesis. (C) 2004 Elsevier Ireland Ltd. All rights reserved.

Differential expression of two Carassius auratus Mx genes in cultured CAB cells induced by grass carp hemorrhage virus and interferon

UV-inactivated GCHV (grass carp hemorrhage virus) is able to induce an antiviral state in cultured CAB cells (crucian carp Carassius auratus blastulae embryonic cells) via the production of interferon (IFN). In the current work, the full-length cDNAs of two Mx genes, termed CaMx1 and CaMx2, have been cloned and sequenced from UV-inactivated GCHV-infected and still IFN-producing CAB cells by suppression subtractive hybridization. Their putative proteins show the characteristically structural features of mammalian IFN-induced Mx proteins, including GTP-binding motif, dynamin family signature and leucine zipper motif. CaMx1 exhibits 85% sequence identity to zebrafish MxA and 72-74% to three Atlantic salmon Mx proteins. CaMx2 is most similar to zebrafish MxE, with 80% identity, and then rainbow trout Mx3, with 52%. Constitutive expression was detected by RT-PCR for CaMx1, but not for CaMx2, in normal CAB cells, but their up-regulations could be induced after treatment with active GCHV, UV-inactivated GCHV and CAB IFN. Distinct kinetics of expression was observed for either CaMx1 or CaMx2 corresponding to the three stimuli, and even between CaMx1 and CaMx2, corresponding to the same stimulus. Upon virus infection, the transcriptional induction was strongly blocked for CaMx2 by cycloheximide (CHX), whereas almost nothing was observed for CaMx1. By contrast, following treatment with CAB IFN, CHX did not inhibit either gene transcription. Collectively, these results suggest that there are very distinct mechanisms for modulating the expression of both CaMx1 and CaMx2 in normal and GCHV-infected CAB cells.

Differential expression and characterization analysis of a new gene with WD domains in fish oogenesis

A new gene with WD domains is cloned and characterized according to its differential transcription and expression between previtellogenic oocytes (phase I oocytes) and fully-grown oocytes (phase V oocytes) from natural gynogenetic silver crucian carp (Carassius auratus gibelio) by using the combinative methods of suppressive subtraction hybridization, SMART cDNA synthesis and RACE-PCR. The full-length cDNA is 1870 bp. Its 5 ' untranslated region is 210 bp, followed by an open reading frame of 990 bp, which has the typical vertebrate initiator codon of ANNATG. The open reading frame encodes a protein with 329 amino acids. It has 670 bp of 3 ' untranslated region and an AATAAA polyadenylation signal. Because it has 92% homology to STRAP (serine-threonine kinase receptor-associated protein), a recently reported gene, we named it FSTRAP (fish STRAP). Virtual Northern blotting indicated that the FSTRAP was transcribed in fully-grown oocytes (phase V oocytes), but not in previtellogenic oocytes (phase I oocytes). RT-PCR analysis showed that FSTRAP was transcribed in brain, heart, kidney, muscle, ovary, spleen and testis, but not in liver. And its mRNA could be detected in the oocytes from phase II to phase V. Western blotting also showed that FSTRAP protein could be detected in brain, heart, kidney, muscle, ovary, spleen and testis except liver. Results of Western blotting on various oocytes were also similar to the RT-PCR data. FSTRAP protein was not expressed in the previtellogenic oocytes. Its expression initiated from phase II oocytes after vitellogenesis, and was consistent with the mRNA transcription.