冷诱导下毛百合甘油-3-磷酸酰基转移酶活性变化及其基因保守区的克隆

探讨了野生毛百合甘油-3-磷酸酰基转移酶活性在冷胁迫下的变化,用简并引物扩增了甘油-3-磷酸酰基转移酶基因保守区序列的结果表明,该保守区段长744 bp,推测编码247个氨基酸.在GPAT氨基酸序列中存在1个高度保守的区域(WIAPSGGRDRP),在NCBI上经过BLASTp比对分析,发现这段保守区序列为LPLAT基...     

土壤微生物DNA木聚糖酶基因多样性的研究

采用国产硅胶Gk254（60型）代替进口Glass bead的改良方法抽提土壤微生物DNA，然后设计了一对扩增木聚糖酶基因片段的新简并引物，对抽提的土壤微生物DNA进行PCR扩增。扩增片段连接pMD18T载体，转化大肠杆菌，重组片段通过酶切进行RFLP分析后，测序分析得到10个木聚糖酶基因片段。对所得片段翻译的氨基酸序列进行BLAST分析表明有8个片段与来自放线菌的木聚糖酶具有较高的同源性，2个与假单胞菌的木聚糖酶具有较高的同源性。所得10个木聚糖酶片段的氨基酸序列同源性比较显示，第27个氨基酸均为天冬酰胺（N），暗示这些来自土壤微生物DNA的基因片段编码耐碱的木聚糖酶。通过构建系统进化树，发现扩增的木聚糖酶片段之间的相似性均在70％以上。     

Diversity of laccase genes from basidiomycetes in a forest soil

Fungal oxidative exo-enzymes lacking substrate specificity play a central role in the cycling of soil organic matter. Due to their broad ecological impact and available knowledge of their gene structure, laccases appeared to be appropriate markers to monitor fungi with this kind of oxidative potential in soils. A degenerate PCR-primer pair Cu1F/Cu2R, specific for basidiomycetes, was designed to assess directly the diversity of laccase genes in soils. PCR amplification of mycelial cultures and fruit-bodies of a wide spectrum of basidiomycetes, covering all functional groups (saprophytes, symbionts, and pathogens), produced multiple DNA fragments around 200 bp. A neighbor-joining tree analysis of the PCR-amplified laccase sequences showed a clear species-specificity, but also revealed that most fungal taxa possess several laccase genes showing a large sequence divergence. This sequence diversity precluded the systematic attribution of amplified laccase of unknown origin to specific taxa. Amplification of laccase sequences from DNA, extracted from a brown (moder) forest soil, showed a specific distribution of laccase genes and of the corresponding fungal species in the various soil horizons (O_h, A_h, B_v). The most organic O_h-horizon displayed the highest gene diversity. Saprophytic fungi appeared to be less widespread through the soil horizons and displayed a higher diversity of laccase genes than the mycorrhizal ones.     

抗稻瘟病菌链霉菌JK-1聚酮合酶合成基因的克隆

利用前人已报道的经典的KA系列、LC系列及MT系列简并引物扩增Streptomyces JK-1基因组DNA,共获得10条目标片段,所有序列的GC含量均在70%左右,具有链霉菌的高GC含量特性。其中片段1与S.coelicolor A3(2)的AL939117.1基因序列在50%可比对序列上有90%的相似性,片段2与S.ambofaciens ATCC 23877的Am238663.1基因序列在95%可比对序列上有81%的相似性,片段10与S.avermitilis MA-4680的BA000030.3基因序列在93%可比对序列上有85%的相似性。其余7条序列与GeneBank数据库中的序列无相似性。分析结果表明,已获得了新的PKS基因的部分片段,这些片段可以作为下一步获得PKS基因全长的有效探针。     

Identification of host plant resistance to pepper leaf curl virus in chilli (Capsicum species)

Three hundred and seven genotypes belonging to four cultivated and one wild species of Capsicum were screened against pepper leaf curl virus (PepLCV) causing devastating leaf curl disease of chilli (Capsicum annuum). Initial screening was done under field conditions based on coefficient of infection (CI), disease reaction to each genotype was assigned. Subsequently, selfed progenies of eight symptom-less and highly resistant lines were challenged by viruliferous white fly under glasshouse conditions, out of which only three genotypes, viz. GKC-29, BS-35 and EC-497636 showed no symptom. Using scion and root stalk of susceptible genotype (Pusa Jwala), these three putative symptom-less genotypes were further challenged by grafting and alternate grafting. The resistant reactions of GKC-29, BS-35, EC-497636 were confirmed because even after 50 days of successful grafting/alternate grafting, no viral symptom appeared on all the grafted plants of these genotypes. When subjected to PCR amplification with degenerate primers deigned to detect gemnivirus like PepLCV, the three symptom-less genotypes did not show any amplification, suggesting that the resistant reaction in three identified symptom-less resistant sources was because of the absence of viral genome and they are not symptom-less carrier.     

利用差异显示技术克隆小麦抗白粉病相关基因的研究

对小麦—簇毛麦抗病易位系进行差异显示分析,以期获得小麦抗白粉病基因的分子克隆。根据已克隆植物抗病基因的保守域,设计了简并引物,与锚定引物组合,对抗病诱导的小麦抗白粉病易位系进行了差异显示分析,共获得10个差异片段,其中两个片段R3-1和8C1.3-6Northern杂交为阳性。将这两个片段克隆后进行了测序,序列分析结果为两个新序列,GenBank登录号分别为AF498271和AF498272。R3-1没有发现同源性较高的植物基因序列,发现8C1.3-6与Sphenostylisstenocarpa 类几丁质酶基因有同源性。     

环状共轭多烯烃简并分子轨道的群论处理

以苯分子为例用群论方法对简并能级进行了处理,得出了用群论方法处理环状共轭多烯烃(CnHn)简并能级分子轨道的一般方法,证明了简并分子轨道的不同表示形式实质上是等价的,介绍了3-8元环状共轭多烯烃的分子轨道。     

简并寡核苷酸引物PCR扩增单细胞基因组的影响因素研究

[目的]研究影响简并寡核苷酸引物聚合酶链反应(degenerate oligonucleotide primed PCR,DOP-PCR)扩增单细胞全基因组的因素。[方法]以单个人外周血淋巴细胞为材料,进行DOP-PCR,研究新鲜细胞与冷藏细胞及采用新鲜和冻存的细胞裂解物为模板对扩增产物的均匀性及特异性的影响。[结果]以新鲜的淋巴细胞为模板与以冷藏的淋巴细胞为模板相比,扩增产物的均匀性相当,特异性上新鲜细胞较好。细胞裂解后立即进行扩增的效果在产物的均匀性及特异性方面均明显优于冻存的细胞裂解物。[结论]对单细胞应用DOP-PCR方法进行全基因组扩增时,应尽量选取新鲜细胞进行裂解,裂解后避免冻存以保证扩增的均匀性及特异性。     

设施杏抗风险栽培研究

设施杏效益高、发展快,但近年的生产现状表明达到丰产要求的设施杏很少.依据各生产因素对设施杏的影响不同,划分为"致死因素"、"非致死因素"、"偶然因素"和"非风险因素"几方面,总结历年生产经验和研究成果,制定了科学的设施杏抗风险栽培的技术规程,为果农生产操作提供借鉴.     

香石竹坏死斑点病毒的鉴定与检测

调查表明云南省昆明市团结乡香石竹普遍发生病毒病。电镜负染检测采自团结乡香石竹病毒病病样,病叶汁液中观察到弯曲长线型的病毒粒体,长度约1000~1600 nm,直径约12 nm。对这些病样的叶片进行间接ELISA检测,所有样品与香石竹坏死斑点病毒抗血清都呈阳性反应。按照报道的长线形病毒属简并引物合成引物,采用RT-PCR法对血清反应呈阳性的香石竹总RNA扩增了1059ntsHSP70基因部分编码序列,将其克隆到pMD18-T载体上,并进行序列分析。根据测序结果设计引物建立RT-PCR扩增检测方法并测定其灵敏度。结果表明,使用简并引物和新设计的引物均能从CNFV感病香石竹组织中扩增出与预期大小一致的目标片段,而健康组织无此扩增产物。灵敏性测定结果表明该特异性引物RT-PCR可从稀释106植物总RNA中检测出病毒。