小麦高分子量谷蛋白亚基的遗传特性研究

利用聚丙烯酰胺凝胶电泳(SDS-PAGE)方法,在蛋白质水平上对多个亲本及其杂交获得的正反交F1代杂交籽粒进行小麦高分子量谷蛋白亚基(HMW-GS)分析,研究其遗传规律.结果表明:小麦高分子量谷蛋白亚基具有品种特性,表达受遗传控制,不受环境影响;小麦高分子量谷蛋白业基在F1代有些组合呈共显性,有些组合呈倾母性的遗传特性.     

Protective immune responses of recombinant outer membrane proteins OmpF and OmpK of Aeromonas hydrophila in European eel (Anguilla anguilla)

Outer membrane proteins (Omps) of Gram‐negative bacteria have been proven to be efficient subunit vaccines against bacteriosis. In this study, OmpF and OmpK of Aeromonas hydrophila were expressed, and their immune protective effects in European eel (Anguilla anguilla) were evaluated. The genomic DNA of A. hydrophila 322A was used as a template, and two kinds of prokaryotic expression plasmids, pET‐32a‐OmpF and pET‐32a‐OmpK, were constructed. Recombinant OmpF protein (r‐OmpF) and r‐OmpK were purified and were proven to have antigenicity by Western‐blot analysis. r‐OmpF and r‐OmpK were used as immunogens to immunize European eel by intraperitoneal injection. The mRNA expression of 6 immune‐related genes (IgM, IL‐10, IRF3, IRF7, LysG4 and HexB) in the liver tissues of eels at 1 hr, 3 hr, 6 hr, 12 hr, 24 hr, 72 hr and 10 days postimmunization was analysed by real‐time PCR. At 30 dpi, the serum antibody response was measured by ELISA. Fish were attacked at 15 dpi by live 322A to assess the protective immunity of r‐OmpF and r‐OmpK. All the six tested genes responded to r‐OmpF or r‐OmpK vaccination at varying degrees. The serum antibody titre of r‐OmpF‐ and r‐OmpK‐immunized groups was 1:1,600 and 1:3,200 respectively. In addition, r‐OmpF gave 35.5% of the relative immune protection rate to European eels, while r‐OmpK gave 70.0%. By analysing the protective immunity and the regulatory role in the immune‐related gene expression of the two recombinant proteins that were studied, it was found that r‐OmpK was a potential vaccine candidate against A. hydrophila.     

基于线粒体COⅠ基因的中国近海棱鳀属鱼类DNA条形码

为明确中国大陆近海棱鳀属鱼类的分类地位,采用国际通用的COⅠ基因5'端652bp序列作为DNA条形码,对中国近海棱鳀属全部6种鱼类62尾标本进行鉴定分析。结果发现,所分析样品的序列碱基组成为T:29.0%,C:26.3%,A:25.3%,G:19.4%,A+T含量(54.3%)高于G+C含量(45.7%),转换/颠换率为3.76。6种棱鳀属鱼类组成5个自展支持率为100%的分支,除黄吻棱鳀和中颌棱鳀各为单系但聚合为一支外,其余4种均独立成支;分支内与分支间平均遗传距离分别为0.2%(0.0%~0.4%)和17.7%(15.7%~19.0%)。赤鼻棱鳀、汉氏棱鳀、杜氏棱鳀和长颌棱鳀均符合Hebert提出的种间遗传距离(15.7%~18.6%)大于或等于10倍种内遗传距离(0.0%~0.3%)的标准,确定了它们的物种有效性。黄吻棱鳀和中颌棱鳀的种内遗传距离皆为0.1%,与其他4种棱鳀的种内遗传距离处于同一水平;但二者种间遗传距离仅为0.6%,明显低于其他物种间的种间遗传距离,属于一般物种的种内遗传距离范围,表明二者亲缘关系很近;由于外部形态存在一定的差异,且在分子系统树上各为单系,二者可作为同一物种的2个不同亚种处理,但也不排除是2个近期分化形成物种的可能,在资源管理上应作为2个不同的进化显著单位分别加以管理。     

菜豆荚斑驳病毒外壳蛋白大小亚基的原核表达及其抗血清制备

本研究对菜豆荚斑驳病毒大、小亚基编码的基因密码子进行了优化并人工合成了这两个基因,然后克隆到原核表达载体p ET-22b(+)中,通过转化大肠杆菌BL21(DE3)菌种并在IPTG的诱导下进行了成功表达,大亚基表达产物分子量为41 k D、小亚基表达产物分子量为22 k D,并制备出了大小亚基基因表达产物的抗血清。测试结果表明,优化后的CP的大、小亚基基因在37℃、1.0 mmol/L IPTG诱导下,3 h后得到了成功的表达,制备的两种抗血清特异性强、效价都达到1∶3.2×10~4,可以对BPMV CP 3个不同的区域同时进行检测,提高了检测的准确度。     

大菱鲆心脏细胞系的建立与鉴定

为丰富海水鱼类细胞系的数量,提供更多的基因功能研究及病毒分离、鉴定工具,本研究建立了一种新的细胞系,大菱鲆心脏细胞系(Scophthalmus maximus heart cell line,SMH).该细胞系使用组织块法(tissue block method)启动原代培养,培养液是添加了胎牛血清(fetal bovine serum,FBS)、人类碱性成纤维生长因子(basic fibroblast growth factor,bFGF)、抗生素、β-巯基乙醇(2-mercaptoethanol,2-ME)、hepes(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)的DMEM/F 12.结果显示,SMH形态呈典型的成纤维细胞样,在培养液中生长分裂旺盛,经230 d传代培养,成功传至58代.用带有绿色荧光蛋白(green fluores-cent protein,GFP)的pEGFP-N3质粒转染SMH,发现GFP在细胞中成功表达,转染效率为40％左右.使用遗传霉素(geneticin,G418)对重组质粒细胞进行筛选,得到了一簇荧光表达效率高的细胞.染色体分析表明,具有正常二倍体核型(2n=44)的细胞占64％.线粒体细胞色素C氧化酶Ⅰ(cytochrome oxidase sub-unit Ⅰ,CO Ⅰ)基因鉴定出该细胞系来源于大菱鲆.该细胞系的建立为大菱鲆功能基因及其他基于细胞系的研究奠定了基础,对大菱鲆的病毒感染途径和分子机制研究具有重要的理论意义.     

禽大肠杆菌病疫苗研究进展

禽致病性大肠杆菌(APEC)是引起家禽多种肠外疾病如气囊炎、心包炎、肝周炎、蜂窝织炎及败血症的主要人畜共患病原菌.在过去的数十年间,试验了多种不同类型的疫苗用于本病的控制.鉴于APEC血清型较多且不同血清型之间缺乏交叉免疫保护,故生产上使用的APEC疫苗研究进展缓慢.作者对不同类型APEC疫苗进行了综述并提出未来的研究方向.     

Effect of allelic variation at the Glu-3/Gli-1 loci on breadmaking quality parameters in hexaploid wheat (Triticum aestivum L.)

Low molecular weight glutenin subunits (LMW-GS) encoded by the Glu-3 loci are known to contribute to wheat breadmaking quality. However, the specific effect of individual Glu-3 alleles is not well understood due to their complex protein banding patterns in SDS-PAGE and tight linkage with gliadins at the Gli-1 locus. Using DNA markers and a backcross program, we developed a set of nine near isogenic lines (NILs) including different Glu-A3/Gli-A1 or Glu-B3/Gli-B1 alleles in the genetic background of the Argentine variety ProINTA Imperial. The nine NILs and the control were evaluated in three different field trials in Argentina. Significant genotype-by-environment interactions were detected for most quality parameters indicating that the effects of the Glu-3/Gli-1 alleles are modulated by environmental differences. None of the NILs showed differences in total flour protein content, but relative changes in the abundance of particular classes of proteins cannot be ruled out. On average, the Glu-A3f, Glu-B3b, Glu-B3g and Glu-B3i_Man alleles were associated with the highest values in gluten strength-related parameters, while Glu-A3e, Glu-B3a and Glu-B3i_Chu were consistently associated with weak gluten and low quality values. The value of different Glu-3/Gli-1 allele combinations to improve breadmaking quality is discussed.     

基于线粒体COⅠ基因序列的三疣梭子蟹东海区群体遗传多样性分析

通过对分布在东海区远海区(济州岛海域)和近海区(长江口以南和以北海域)的三疣梭子蟹(Portunus trituberculatus)自然群体线粒体COⅠ基因部分序列的测定,比较并分析了其群体遗传多样性和遗传结构。在24个采集点共286个样本的线粒体COⅠ序列中共检测到45种单倍型,单倍型多样性为0.864;核苷酸多样性为0.002 84,平均核苷酸变异数是1.686,其中有23个单倍型为单个采集点所独享,Hap3出现频率最高,在23个采样点的83个样本中均有发现,是所有三疣梭子蟹样本的共享中心单倍型。群体遗传多样性分析显示,远海区群体的遗传多样性水平最高,其次为长江口以北群体,长江口以南群体最低。由群体间遗传距离、系统进化树和单倍型网络关系图发现,东海区海域三疣梭子蟹种群间存在基因交流,亲缘关系的远近不以地理位置的远近为依据;AMOVA分子方差分析结果显示,三疣梭子蟹的遗传变异主要来自群体内,而群体间无显著遗传分化,不存在明显的地理趋势。     

Marker‐assisted backcrossing of a null allele of the α‐subunit of Soybean (Glycine max) β‐conglycinin with a Chinese soybean cultivar (a). The development of improved lines

The development of soybean varieties that lack the β‐conglycinin α‐subunit is an attractive goal because the β‐conglycinin α‐subunit negatively influences the nutrition and gelation of tofu and is a major allergen. To remove this undesirable allergen and simultaneously improve the seed nutritional value and food‐processing quality, marker‐assisted background selection (MABS) was used in backcross breeding to incorporate cgy‐2, a null phenotype version of the gene encoding the β‐conglycinin α‐subunit, from the donor line ‘RiB’ into the genetic background of the Chinese cultivar ‘Dongnong47’ (DN47), a popular high‐oil superfine seed soybean cultivar from Heilongjiang Province, China. In each F₂ (F₂, BC_nF₂) generation of the breeding programme, the offspring that carried the introgressed cgy‐2 were identified by sodium dodecyl sulphate–polyacrylamide gel electrophoresis and rescreened by MABS using simple sequence repeat markers to accelerate recurrent parent genome recovery. Of the 49 advanced backcrossing breeding lines (ABLs), the three best lines, ABL1, ABL2 and ABL3, were selected from the BC₁, BC₂ and BC₃ populations, respectively. The ABLs were evaluated for desirable agronomic characteristics, yield‐related traits, amino acid composition, free amino acid composition and tofu‐processing quality in the mature seeds. All of the ABLs lacked the α‐subunit but grew and reproduced normally without deleterious effects on physiological processes such as seed development and germination. The free amino acid content of ABL1 was significantly higher than that of ‘DN47’, with arginine (Arg) being particularly enriched. Compared to the recurrent parent ‘DN47’, the total protein content of the three ABLs was higher, the amino acid composition of the seed proteins was markedly modified and the yield and hardness of the tofu that was made from the ABLs were significantly increased. MABS combined with stringent phenotypic selection in a backcross breeding programme is a feasible strategy for the genetic engineering of seed protein components to produce allergenic subunit‐deficient variant alleles.