马疱疹病毒1型主要毒力基因分析及TK基因缺失载体的构建

为了解新疆马疱疹病毒1型（EHV-1）主要毒力基因遗传进化情况并构建TK基因缺失株，本研究以EHV-1 XJ2015株DNA为模板，对其主要毒力基因TK、gI和gE全长进行克隆、测序及生物信息学分析，并扩增TK基因左右重组臂TKL和TKR，构建质粒pUC-TKLR，将扩增后的增强绿色荧光蛋白（EGFP，含有CMV+polyA）插入pUC-TKLR质粒，构建TK基因缺失打靶质粒。TK、gI和gE基因同源性分析结果显示，XJ2015株与国外EHV-1分离株TK、gI和gE基因同源性均较高，分别为99.8%~100.0%、99.6%~100.0%和99.9%~100.0%；与EHV-3分离株同源性均最低，分别为72.9%、59.4%和62.1%；遗传进化分析显示，3个基因均与国外EHV-1同属于一个遗传进化分支，与EHV-9和EHV-4进化关系较近，但与EHV-3进化关系较远，表明XJ2015毒株与国外EHV-1毒株TK、gI、gE基因核苷酸上差异不明显，没有明显的地域性特征，功能基因保守且进化缓慢，同源基因功能相同或相近；经PCR扩增、酶切、测序及转染鉴定，本试验成功构建了用于TK基因缺失的打靶质粒pUC-TKLR-EGFP。通过对EHV-1主要毒力基因的分析及TK基因缺失打靶载体的构建，为新疆地区马鼻肺炎流行病学调查分析、TK基因缺失株的构建提供理论依据。     

Assessment of the ideal ratios of digestible essential amino acids for pacu,Piaractus mesopotamicus,juveniles by the amino acid deletion method

The present study aimed to determine the ideal ratios of digestible essential amino acids (EAAs) for pacu (Piaractus mesopotamicus) juveniles by the amino acid (AA) deletion method. A completely randomized design which consisted of 11 treatments and three replicates each was used. The treatments included a control diet (CD) containing 55% of nonpurified natural ingredients and 45% of purified synthetic amino acids and ingredients, and other ten isonitrogenous and isoenergetic EAA limiting diets (LDs), each being deficient in 44.4 ± 0.02% of the respective EAA. Pacu juveniles with initial average body weight of 6.22 ± 0.09 g were distributed among 33 fiber glass tanks. Fish were fed with semipurified and extruded diets for 113 days two times a day until apparent satiation. The ideal ratio of each dietary EAA was calculated on the basis of the relationship between body N retention and amount of EAA deleted from the respective EAA LD. Based on the AA deletion method, the ideal ratios of digestible EAAs for pacu juveniles, relative to lysine requirement of 100% were estimated as: methionine 14.6%, threonine 35.0%, tryptophan 6.6%, arginine 62.8%, histidine 13.6%, isoleucine 26.3%, leucine 43.7%, phenylalanine 27.2%, and valine 35.8%.     

Utilization of deletions to localize a gene for resistance to the cereal cyst nematode, Heterodera avenue, on an Aegilops ventricosa chromosome

A previous RFLP analysis showed that the Aegilops ventricosa chromosome 6M^V which compensates for the absence of 6D in 6M^V (6D) wheat substitution lines was a 2/6 translocated chromosome, either 2S–6S.6L or 2S–6L.6S. The distal part of its long arm consists of a translocated segment belonging to homoeologous group 2. Chromosome 6M^V carries a gene(s) for resistance to cereal cyst nematode. In order to define the part of 6M^V (2S or 6S or 6L) involved in this resistance, addition lines with a 6M^V deleted either for its short arm or for the distal part of its long arm were evaluated. It was shown that the gene(s) is carried by the group 2 translocated segment. The hypothesis that the gene(s) could be allelic to Cre2, another gene conferring resistance to the nematode introduced into the wheat complement from Ae. ventricosa is discussed.     

Identification and breeding behaviour of a major deletion of chromosome 5D ofTriticum aestivum

Summary A study was undertaken to evaluate the breeding behaviour and to identify a spontaneously produced putative chromosomal deletion in the winter wheat (Triticum aestivum L. em. Thell.) cv Norstar. Male and female transmission studies of plants heterozygous for the deletion chromosome indicated 9.5% and 48.8% transmission through the pollen and the egg, respectively. Meiotic analyses of progeny from deletion heterozygotes indicated that the deletion chromosome was eliminated from half of the plants, which agreed with the male and female transmission frequencies. Testcrosses of the deletion chromosome with telocentrics and nullisomic-tetrasomic combinations suggested that the deletion involved the long arm of chromosome 5D. This was confirmed by restriction fragment length polymorphism (RFLP) analysis. Also, monosomic plants obtained in progeny of deletion heterozygotes were shown to be monosomic for 5D. Studies of plants homozygous for the deletion showed relatively normal pairing between the deletion chromosomes, and with the short arm (5DS), but not the long arm (5DL). Deletion homozygotes were self-sterile, and morphologically similar to plants nullisomic for 5D, but plants that also contained 5DL, or a homoeologous chromosome were self-fertile and had normal morphology. Studies of chromosome morphology indicated that the deletion chromosome was metacentric, and the length of the long arm was reduced by approximately 60%. RFLP studies showed that, in terms of genetic distance, 90% of the arm was missing.     

The Use of Multivariate Analyses to Eliminate Redundant Variables when Evaluating Potatoes for Chip Quality

Selection for good chipping clones in potato breeding often takes place during the middle and late selection stages. This is due to the considerable resources needed for the assessment of several characteristics. Objective multivariate methods for the elimination of redundant variables were applied to eight standardized chipping quality traits recorded on advanced clones over 15 years at two locations in New Brunswick, Canada. The clones were evaluated of specific gravity and the following chipping scores: direct from the field in August; after 10 days storage at 13 °C; from 4 °C storage in February; from 4 °C storage in February with 1 week, 2 weeks, 3 weeks and 4 weeks reconditioning at 16 °C. Five elimination methods produced a similar deletion order. In every subset obtained, specific gravity and direct chipping from the field were always retained. Reconditioning treatments from one to four weeks after cold storage were shown to be the most redundant. The reduction in the number of traits from eight to four by every technique could recover more than 80 % of the original variation, showing that redundant variables were effectively discarded.     

山羊痘病毒SS株5个ANK基因缺失的重组病毒构建

为研究锚定蛋白基因(Ankyrin,ANK)对山羊痘病毒的影响,试验采用融合PCR和Overlap PCR技术扩增山羊痘病毒SS株5个ANK基因(ANK010、ANK138、ANK140、ANK141.2和ANK145)两端侧翼序列和绿色荧光蛋白(GFP)基因,并将其产物连接Trans1-T 1载体构建ANK缺失的转移载体,经过菌液PCR以及质粒双酶切鉴定,阳性重组质粒用Lip 2000转染至已经感染羊痘病毒SS株的羊睾丸原代细胞,依报告基因GFP的表达情况在荧光显微镜下筛选目的基因缺失的重组病毒,同时设立不感染病毒的对照。结果表明:通过融合PCR方法成功扩增山羊痘病毒SS株ANK基因010和138的两端侧翼序列及GFP基因片段,大小约1200 bp;通过Overlap PCR方法成功得到ANK基因140、141.2和145基因的侧翼及GFP基因片段,大小约900 bp,与理论相符。研究成功构建了基因缺失转移载体,将其转染感染SS病毒的细胞中,5个ANK基因缺失的表达载体均可见绿色荧光斑点,说明得到各自基因缺失的重组羊痘病毒。     

尖孢镰刀菌番茄专化型中SNARE蛋白FolSso1的功能分析

 SNARE(Soluble N-ethylmaleimide-sensitive factor attachment protein receptor)蛋白保守存在于丝状真菌中,在膜泡转运的过程中起着关键的作用。番茄枯萎病是由尖孢镰刀菌番茄专化型(Fusarium oxysporum f. sp. lycopersici,Fol)引起的,严重威胁着番茄的生产。我们使用反向遗传学的方法来研究番茄枯萎病菌中SNARE蛋白FolSso1的功能,实验结果发现FolSSO1的基因缺失突变体菌丝生长速率降低,且产孢数量减少。另外,FolSSO1基因的缺失导致突变体相较于野生型菌株对细胞壁压力与细胞膜压力更加敏感。然后,在番茄果实和番茄植株的致病性实验中,我们发现FolSSO1的缺失并没有引起Fol致病性显著的变化。综上所述,本研究发现FolSSO1可以调控Fol营养生长,繁殖和对环境压力的响应过程,然而对Fol的致病过程并没有显著的调控作用。     

鸭肠炎病毒UL41基因缺失株的构建及其体外增殖能力分析

旨在构建鸭肠炎病毒（DEV）UL41基因缺失毒株,并对其生物学特性进行分析,本研究以克隆有DEV UL41基因的重组黏粒D1为骨架,利用Red/ET重组技术构建缺失UL41基因的重组黏粒D1 dUL41;将UL41基因缺失重组黏粒D1 dUL41与其他4个克隆有DEV基因组片段的亲本黏粒共转染鸭胚成纤维细胞（DEF）,拯救获得UL41基因缺失毒株rDEV-SD19/dUL41。将该基因缺失病毒感染DEF后,提取病毒基因组进行PCR鉴定及测序,并利用间接免疫荧光试验检测该病毒感染细胞中UL41基因表达情况;绘制拯救的基因缺失病毒的生长曲线,分析其体外复制特性。PCR及测序结果显示,本研究成功构建了缺失UL41基因的重组黏粒D1 dUL41。将该基因缺失黏粒与其他含有DEV基因组的亲本黏粒共转染DEF后能够产生典型的蚀斑病变。PCR及测序结果显示,UL41基因成功从DEV基因组中缺失;间接免疫荧光试验发现,基因缺失病毒rDEV-SD19/dUL41感染DEF后,未见UL41蛋白表达。综上表明,本研究成功构建了DEV UL41基因缺失病毒。体外生长曲线显示,rDEV-SD19/dUL41在DEF中的复制能力明显低于亲本病毒,提示UL41蛋白在DEV复制中发挥重要作用。UL41基因缺失DEV的构建为进一步研究UL41基因在DEV感染和致病中的作用机制奠定了基础。     

猪布鲁菌S2omp10基因缺失株的构建及特性

现有的布鲁菌减毒活疫苗存在一定毒力,且野强毒株和减毒活疫苗株间缺少可供鉴别的抗原,导致在血清学检测上自然感染与疫苗接种很难区分,限制了现有的减毒活疫苗的广泛应用.本文拟对布鲁菌的减毒活疫苗株S2进行遗传改造,克服上述缺陷.本研究利用同源重组的方法,得到了布鲁菌S2株omp10基因缺失株.分别用基因缺失株和疫苗株感染小鼠,比较基因缺失株小鼠体内的存活能力.结果成功构建了布鲁菌S2株omp10基因缺失株,动物试验结果表明,基因缺失株仍能在小鼠体内存活,具备作为减毒活疫苗的特性.与原始S2株比较,基因缺失株的感染力进一步减弱.表明omp10基因在布鲁菌的毒力及体内生存方面发挥了作用,为基因标记疫苗的研制奠定了基础.     

布鲁菌缺失疫苗株M5-ΔznuA和M5-Δbp26-ΔznuA的构建及毒力和免疫原性的评估

【目的】利用分子生物学技术,通过同源重组的方法构建了新型的羊种布鲁菌Brucella melitensis减毒活疫苗株,为布鲁菌的防治提供新的选择.【方法】以羊种布鲁菌减毒活疫苗M5株及缺失bp26基因的M5-Δbp26缺失突变株为亲本株,利用电击转化法,将含有znuA基因上下游同源臂的自杀质粒转入到亲本株中,通过同源重组敲除znuA基因.对构建的新型基因缺失株进行生物学特性及毒力的鉴定与分析.【结果和结论】PCR及核苷酸序列测序结果表明,羊种布鲁菌减毒活疫苗znuA单基因缺失株和bp26、znuA双基因缺失株均构建成功,分别将其命名为M5-ΔznuA和M5-Δbp26-ΔznuA.与亲本株相比,2种缺失突变株的生物学特性没有显著性差异;体外连续传至20代后,菌落PCR和核苷酸测序结果显示M5-ΔznuA和M5-Δbp26-ΔznuA株具有良好的遗传稳定性.小鼠脾脏质量和脾脏菌落数表明,M5-Δbp26-ΔznuA双基因缺失株毒力最弱,单基因缺失株M5-ΔznuA和M5-Δbp26次之.血清中抗体水平的监测显示,znuA基因的缺失对布鲁菌减毒活疫苗诱导机体体液免疫的能力没有影响.获得了免疫原性保持良好而毒力减弱的羊种布鲁菌减毒活疫苗基因突变株M5-ΔznuA和M5-Δbp26-ΔznuA.