果梅PmKNAT2基因全长cDNA克隆及表达分析

【目的】从‘大嵌蒂’果梅中克隆PmKNAT2,并对其结构特征及表达模式进行分析,为研究果梅雌蕊败育的分子机理和分子育种奠定基础。【方法】在NCBI中查找与果梅相似性很高的桃KNOPE2（KNAT2的同源基因）序列（EF093491）,根据桃的KNOPE2序列设计特异引物;采用改良CTAB法提取‘大嵌蒂’果梅花芽总RNA;通过RT-PCR和RACE技术获得基因cDNA序列全长;使用DNAMAN软件进行序列拼接;利用NCBI数据库中的BLASTn和BLASTp程序进行相似性分析;通过生物信息学方法分析结构特征;用DNAMAN软件分析基因的ORF和氨基酸序列;MEGA4.0软件进行系统进化树的构建;利用Bioxm2.6推测分析蛋白分子量和等电点;根据NCBI中Conserved Domains程序进行蛋白质保守域结构预测;用ExPaSy提供的在线SOPMA程序进行蛋白质二级结构预测;构建PJIT166-PmKNAT2-GFP的融合亚细胞定位表达载体,采用基因枪法转化洋葱表皮细胞,经暗培养后在激光共聚焦显微镜下观察;利用实时荧光定量RT-PCR研究其表达模式,分析其在‘大嵌蒂’花芽发育不同时期、不同器官中的表达特性。【结果】在‘大嵌蒂’果梅中获得一个KNAT2的同源基因,命名为PmKNAT2,其cDNA全长为1 402 bp,5'UTR 47 bp,3'UTR 293 bp,编码区全长为1 062 bp,编码353个氨基酸,预测蛋白质分子量为40.41 kD,理论等电点为4.85。蛋白结构分析表明该基因编码的氨基酸具有2个结构域,MEINOX结构域（KNOXⅠ和KNOXⅡ 2个亚结构域）和HD（homomeodomain）结构域,属于KNOX蛋白;相似性分析发现该基因与GenBank中其它来源的KNOX蛋白序列的相似性为50%-100%;系统进化树分析显示果梅PmKNAT2与桃KNOX聚为一类,表明与其亲缘关系最近。二级结构预测结果表明PmKNAT2所编码蛋白由47.14%的α-螺旋（Alpha helix）、3.43%的β-转角（Beta turn）、3.14的β-折叠（Extended strand ）和46.29%的随机卷曲（Random coil）组成。亚细胞定位结果显示该基因编码的蛋白定位于细胞核和细胞膜中。实时荧光定量RT-PCR分析表明,在‘大嵌蒂’果梅不同时期花芽中PmKNAT2的表达量存在一定差异,PmKNAT2在11月份的表达量最高,9月、10月和11月的表达量差异不明显,但与12月和1月花芽中的表达量差异明显;生长素在1月份含量最高,9、10、11月份差异不明显,其含量变化趋势与PmKNAT2表达趋势相反。对该基因表达量最高的11月份的花芽进行实时荧光定量分析发现：PmKNAT2在各种花芽中均有表达,在不完全花（雌蕊褐色、雌蕊畸形和无雌蕊）中的表达量高于完全花（雌蕊正常）。进一步分析PmKNAT2在完全花与不完全花的不同花器官中的表达量,结果表明PmKNAT2在完全花与不完全花的各部位的表达量趋势相似均为：萼片>雄蕊>花瓣,在完全花雌蕊中的表达量最低。【结论】推测PmKNAT2在雌蕊发育阶段的异常表达可能与‘大嵌蒂’果梅雌蕊败育有关。     

Equine amnionitis and fetal loss: The case definition for an unrecognised cause of abortion in mares

A series of abortions occurred in mares in New South Wales during 2004 that involved similar and unusual findings on post mortem examination of aborted fetuses and fetal membranes. The term Equine Amnionitis and Fetal Loss (EAFL) was developed to describe the condition. This form of abortion had not been previously recognised in Australia. The pathology alone is not specific for EAFL and diagnosis requires demonstration of a combination of certain pathological and bacteriological features. The purpose of this paper is to describe patterns considered consistent with EAFL cases as a working case definition for use by veterinarians and veterinary pathologists in identifying future cases of EAFL. More detailed papers are in preparation to fully describe the epidemiological, histopathological, and microbiological aspects of EAFL.     

春夏不同播期对玉米单倍体自然加倍率的影响

研究19个玉米单倍体在春、夏播不同播种期对自然加倍率(显花药率、结实株率)的影响。结果表明,不同播期和不同材料对玉米单倍体自然加倍率有明显差异,显花药率最高为52.6%,最低为8.4%,平均为23.3%;结实株率最高为11.2%,最低为0.5%,平均为2.8%。19个材料的单倍体结实率春播时期明显高于夏播时期,16个材料的显花药率表现为春播时期高于夏播时期,春播和夏播玉米单倍体平均显花药率分别为26.9%和19.7%,平均结实株率分别为3.5%和2.1%,表明春播时期更利于玉米单倍体的自然加倍。     

蓖麻花药愈伤组织诱导的研究

以通蓖5号为试验材料,诱导蓖麻花药的愈伤组织.分别以接种密度(1/5个、2/5个、3/5个、4/5个、1个花蕾)、基本培养基种类(MS、NB、B_5、N_6)、低温预处理时间(0d、1d、3d、5d、7d、9d)、培养基添加物(糖、生长激素、脯氨酸、水解酪蛋白、抗坏血酸)等因素为变量,对蓖麻花药愈伤组织进行诱导试验.结果表明,3/5个花蕾的接种密度、30g/L的糖浓度、MS培养基(添加1.5mg/L 6.BA和0.9mg/L NAA)以及5d的低温(4℃)预处理均有利于花药愈伤组织的形成;同时脯氨酸、水解酪蛋白、抗坏血酸等的加入均可有效地提高蓖麻花药愈伤组织诱导率.     

6种枸杞植物花药培养单倍体的诱导

[目的]获得6种枸杞花粉植物。[方法]以6种枸杞花药为材料,在含不同激素的9种MS培养基上分别进行光照和黑暗离体培养。[结果]6种材料都诱导出了愈伤组织,最高愈伤组织诱导率可达20％,愈伤组织转入培养基（MS＋6-BA0．5mg／L＋NAA0．1mg／L）分化出大量绿色小芽,转入生根培养基（MS＋NAA0．1mg／L）中,20d后得到完整植株。[结论]材料不同,在同一培养基上的诱导率不同,在光照培养下愈伤组织诱导率比暗培养条件下的诱导率高。     

假单性结实无核葡萄胚败育时期的研究

以布朗无核和无核白2个假单性结实无核品种为试材,对其胚和胚乳的发育进行细胞学观察．结果表明,无核白和布朗无核胚乳败育的时期分别为授粉后第32天和34天;无核白和布朗无核胚败育的时期分别为授粉后第38天和42天．     

影响刺梨花药愈伤组织诱导因素的研究

以贵农1号、2号、5号、6号、7号刺梨为材料,研究不同基因型、不同取材时间、小孢子发育时期、基本培养基、糖源、生长调节物质、培养条件等刺梨花药愈伤组织诱导的影响.结果表明;以刺梨盛花期(5月份)且小孢子发育为单核中后期的贵农1号花药为试材;Ms为基本培养基;3%的蔗糖为糖源;1.0mg·L-1 6-BA +2.0mg·L-1 NAA +0.5mg·L-1 2、4-D +1.0mg·L-1 KT为激素组合,在黑暗条件下培养最有利于刺梨花药愈伤组织的诱导.     

巴西橡胶树花药体细胞胚形成中乳管分化的研究

培育数量多、质量好的体细胞胚是当前巴西橡胶树花药组织培养的主要研究目标,而对于体细胞胚发育过程中是否有乳管分化目前尚不清楚。以不同发育阶段的正常体细胞胚以及不同形态的畸形胚为材料,对它们的乳管分化情况进行了研究。结果表明,球形胚、心形胚和鱼雷胚中都没有乳管分化,最早出现可辨识的乳管是早期子叶胚,而不同形态的畸形胚都有乳管分化。本研究为深入研究乳管分化的机制提供了基础。     

应用花药培养提纯粳稻不育系XG111A和保持系XG111B

应用花药培养技术对粳稻BT型不育系XG111A和保持系XG111B进行提纯改良。结果表明,低温6～8℃预处理3～18 d,有助于花药愈伤诱导率的提高,尤以低温预处理8 d,愈伤诱导率最高;35～50 d诱导愈伤的分化率最高,XG111A和XG111B的分化率分别达到22.5%和30.2%。经花药培养后,不育系和保持系株系内整齐度均得到显著提高:不育系的不育花粉率为100%,原供体对照为99.58%。保持系的花培后代植株90%以上都具有原供体植株的典型特征,且后代的性状遗传稳定,另有部分株系优于原供体植株。     

Post-flowering leaflet removals increase pod initiation in soybean canopies

Much of the yield variation in soybean (Glycine max L. Merrill) crops is related to changes in pod and seed number. Pod number is the result of pod initiation and pod abscission while seed number is the result of potential seed per pod and seed abortion. However, the physiological regulation of these processes is not well understood. A field experiment was conducted to investigate the role of post-flowering changes in source size and canopy structure on pod initiation, pod abscission and seed abortion in soybean. Two soybean genotypes: DM48 and A7409 (maturity groups IV and VII, respectively) were used. Leaflet removal treatments (L) consisted of removing none (L0), one (L1) or two (L2) lateral leaflets of every developed trifoliate leaf present. Leaflet removals were applied twice: the first at full bloom and the second shortly after the beginning seed stage. Crop growth rate (CGR), leaf area index (LAI), light interception (LI), and relative leaf growth rate, were determined during the periods in which numerical components are established. For the period between the first and the second leaflet removal, CGR remained unchanged among L treatments in both genotypes because LAI reductions were compensated through an increase in the net assimilation rate of the remaining leaves. The first leaflet removal increased the relative leaf growth rate and the number of pods initiated (PI) and these increases were inversely related to the remaining LAI in both genotypes. Moreover, the inverse relationship between LAI and PI was sustained at LAI below and above critical (i.e., LAI for 95% LI) and was not related to CGR or LI. The number of pod abscised also increased with the level of leaflet removal during the first and main abscission period in both genotypes and the percentage of pod abscission was directly related to the seed growth rate per unit leaf area during the abscission period. Seed abortion was inversely related to LAI after the second leaflet removal. Only the highest level of leaflet removals (i.e., L2) was able to reduce seed size in both genotypes. Whereas pod abscission, seed abortion and seed size could be related to indicators of canopy assimilatory capability pod initiation was not, suggesting that other physiological mechanism/s operate in the regulation of pod initiation. In addition, our results suggest that early (i.e., at flowering) canopy closure may negatively impact pod initiation in soybean. To the best of our knowledge, this study is the first to document that the number of initiated pods is inversely related to LAI in soybean canopies.