副猪嗜血杆菌Flp-FRT双基因敲除方法的建立与优化

本研究旨在利用Flp-FRT位点特异性重组系统建立副猪嗜血杆菌(Glaesserella p arasuis)的无抗性标记基因敲除方法,为G.parasuis的毒力因子、致病机制和基因工程疫苗研究提供有力的遗传操作工具.首先利用λ噬菌体cI857/PRM/PR基因表达调控系统调控Flp重组酶的表达,实现抗性突变体中抗性...     

雌蕊缺失茶树花3个发育期的数字基因表达谱分析

以雌蕊缺失茶树花为试材,利用转录组、数字基因表达谱技术,研究了雌蕊缺失茶树花的花芽、花蕾、花3个时期相关基因的表达规律。结果发现,雌蕊缺失茶树花生长发育过程中进行着各种旺盛的生物合成和代谢活动。生长素信号转导途径的6个基因和ABCDE类识别基因的A类、C类和E类可能与茶树花的雌蕊缺失和雄蕊发育密切相关,并且基因调控过程较复杂;WUS基因中WUS2和WUS8下调可能调控了C类和E类基因,从而导致雌蕊的缺失;KNOX家族中,未检测到KNOXⅡ的同源基因,KNOX I类同源基因下调表达和缺失可能减弱了茶树花雌蕊心皮的启动和雌蕊边缘组织的生长。可以看出,本研究通过数字基因表达谱分析,初步了解了雌蕊缺失茶树花发育前后的网络途径,为后期对茶树花雌蕊缺失和雄蕊发育的研究,探明茶树花不育和性别决定基因的分子机制提供理论依据。     

Genetic Characterization of Indigenous Rice Varieties in Eastern Himalayan Region of Northeast India

The eastern Himalayan region of Northeast （NE） India is home to a large number of indigenous rice varieties, which are traditionally classified as Oryza sativa subspecies indica, japonica or intermediate types. The classification based on traditional Cheng’s index is often inconclusive due to phenotypic plasticity of morphological characters, which are influenced by environmental conditions. We used molecular markers specific for indica and japonica subspecies to assess the degree of genetic relatedness of indigenous rice varieties in NE India. The results revealed that majority of upland （jum） and glutinous rice varieties, traditionally considered as japonica, were genetically close to the subspecies indica. All varieties of boro ecotype were found to be indica type, and only a few varieties cultivated in lowland and upland areas were japonica type. Some of the lowland varieties of the sali ecotype were intermediate between indica and japonica, and they showed a closer genetic affinity to O. rufipogon.     

水稻胚乳特异表达启动子DXCP35的克隆及功能鉴定

根据水稻全生育期基因表达谱芯片数据库找到1个在水稻胚乳特异表达的基因,命名为DX35,用PCR技术从水稻品种明恢63基因组中克隆得到其上游1 356bp长度的启动子DXCP35。将DXCP35与β-glucuronidas(GUS)报告基因融合后,经根癌农杆菌(Agrobacterium tumefaciens)介导转化获得转基因水稻阳性植株,通过组织化学染色法证明DXCP35是1个胚乳特异表达启动子。对其进行5′端缺失分析,构建了6个缺失载体,通过验证缺失载体的表达谱,证明308bp长度的启动子就足以维持胚乳特异表达模式。     

Molecular characterization of avian polyomavirus isolated from psittacine birds based on the whole genome sequence analysis

Seven avian polyomaviruses (APVs) were isolated from seven psittacine birds of four species. Their whole genome sequences were genetically analyzed. Comparing with the sequence of BFDV1 strain, nucleotide substitutions in the sequences of seven APV isolates were found at 63 loci and a high level of conservation of amino acid sequence in each viral protein (VP1, VP2, VP3, VP4, and t/T antigen) was predicted. An A-to-T nucleotide substitution was observed in non-control region of all seven APV sequences in comparison with BFDV1 strain. Two C-to-T nucleotide substitutions were also detected in non-coding regions of one isolate. A phylogenetic analysis of the whole genome sequences indicated that the sequences from the same species of bird were closely related. APV has been reported to have distinct tropism for cell cultures of various avian species. The present study indicated that a single amino acid substitution at position 221 in VP2 was essential for propagating in chicken embryonic fibroblast culture and this substitution was promoted by propagation on budgerigar embryonic fibroblast culture. For two isolates, three serial amino acids appeared to be deleted in VP4. However, this deletion had little effect on virus propagation.     

猪繁殖与呼吸综合征病毒强毒株HUB2株全基因组序列分析

从湖北省暴发猪"高热病"的猪场分离出1株猪繁殖与呼吸综合征病毒(Porcine reproductive and respiratory syndrome virus,PRRSV),并命名为HUB2株.根据GenBank上已发表的PRRSV全基因序列设计引物进行RTPCR扩增,获得PRRSV HUB2株全基因组cDNA序列.测序结果表明PRRSV HUB2株基因组全长15 320 bp(不包括PolyA尾).分析结果显示该毒株与PRRSV美洲型标准株(VR-2332)和欧洲型标准株(LV)全基因核苷酸同源性分别为89.6%和50.3%.说明HUB2属于美洲型毒株.与VR-2332相比,HUB2株非结构蛋白(Nsp2)存在2处不连续的缺失(共缺失30个氨基酸),其缺失位点位于推定氨基酸序列的第481位和532～560位.此次新出现的强毒株全基因组序列特性的揭示为科学防治猪高致病性蓝耳病奠定了理论基础.     

外膜蛋白OmpA在蛙源米尔伊丽莎白菌致病性中的功能

为探究外膜蛋白A（outer membrane protein A，OmpA）对米尔伊丽莎白菌致病作用的影响，以蛙源米尔伊丽莎白菌FL160902为研究对象，通过同源重组法构建OmpA缺失株△ompA，比较缺失株和野生株的生长特性、生物膜形成能力、抗血清杀伤能力、对细胞的黏附能力以及对蛙的致病性差异。结果显示：△ompA的生长能力和抗血清杀伤能力与野生株无显著差异；但与野生株相比，△ompA的生物膜形成能力增加了66%，△ompA对bEnd.3细胞的黏附能力降低了61%；黑斑蛙感染试验显示，△ompA在黑斑蛙血液、脾和脑组织中的载菌量分别为（3.15×10⁸±0.09×10⁸）、（2.11×10⁸±0.07×10⁸）和（6.61×10⁸±0.16×10⁸） copies/g，均显著低于野生株，且△ompA对黑斑蛙的致死率为37%，显著低于野生株的致死率（75%）。上述结果表明，ompA基因缺失不改变米尔伊丽莎白菌的抗血清杀伤能力，但增加了菌株的生物膜形成能力，减弱了菌株的黏附能力，从而降低了该菌对蛙的致病性。     

Methods of Determining and Deleting Additional Dummy Arrows in Network Diagram

Based on analysis of the function of dummy arrow in network diagram, this paper provides some methods to determine and delete additional arrows. It will be helpful to personnel in engineering network planning for determining and deleting the additional dummy arrows quickly and correctly.     

Molecular and physical mapping of genes affecting awning in wheat

Quantitative trait loci (QTL) for three traits related to awning (awn length at the base, the middle and the top of the ear) in wheat were mapped in a doubled‐haploid line (DH) population derived from the cross between the cultivars ‘Courtot’ (awned) and ‘Chinese Spring’ (awnless) and grown in Clermont‐Ferrand, France, under natural field conditions. A molecular marker linkage map of this cross that was previously constructed based on 187 DH lines and 550 markers was used for the QTL mapping. The genome was well covered (more than 95%) and a set of anchor loci regularly spaced (one marker every 20.8 cM) was chosen for marker regression analysis. For each trait, only two consistent QTL were identified with individual effects ranging from 8.5 to 45.9% of the total phenotypic variation. These two QTL cosegregated with the genes Hd on chromosome 4A and B2 on chromosome 6B, which are known to inhibit awning. The results were confirmed using ‘Chinese Spring’ deletion lines of these two chromosomes, which have awned spikes, while ‘Chinese Spring’ is usually awnless. No quantitative trait locus was detected on chromosome 5A where the B1 awn‐inhibitor gene is located, suggesting that both ‘Courtot’ and ‘Chinese Spring’ have the same allelic constitution at this locus. The occurrence of awned speltoid spikes on the deletion lines of this chromosome suggests that ‘Chinese Spring’ and ‘Courtot’ have the dominant B1 allele, indicating that B1 alone has insufficient effect to induce complete awn inhibition.     

Mapping of chlorina mutant genes on the long arm of homoeologous group 7 chromosomes in common wheat with partial deletion lines

The chlorophyll a:b ratio of chlorina mutants is much higher than that of wild type plants. Physical mapping of the chlorina mutant loci (cn-A1, cn-B1 and cn-D1) of common wheat (Triticum aestivum L.) and durum wheat (T. turgidum L.) was carried out with partial deletion lines of Chinese Spring(CS) of the long arms of homoeologous group7 chromosomes. F₁ plants of partial deletion lines with near-isogenic lines (ANK-32A and ANK-32B) of the spring bread wheat Novosibirskaya 67 and a near-isogenicline of durum wheat LD222, ANW-7B were evaluated for chlorophyll a:b ratio of the leaves. Hemizygous and heterozygous plants were more easily distinguished by chlorophyll a:b ratio than by visual observation. The dose effects of the chlorina loci on chlorophyll a:b ratio were also confirmed. The position of the allele on the chromosome was localized by fraction length, the comparative values between whole chromosome and partially deleted chromosome. The locus cn-A1 was localized on the region of 83% distal from the centromere on the long arm of chromosome 7A, cn-B1 locus was localized on the region between 69% and 78% distal from the centromere on the long arm of chromosome 7B, and cn-D1 locus was localized on the region between 76% and 77% distal from the centromere on the long arm of chromosome 7D. We consider the map derived by deletion mapping is more accurate than the map calculated from recombination frequency. This revised version was published online in July 2006 with corrections to the Cover Date.