P-loop，Bet v 1结构域的缺失及突变对GmPR10 和Gly m 4l 抑制大豆疫霉菌能力的影响

大豆疫霉根腐病是由大豆疫霉菌（Phytophthora sojae）引起的危害大豆生长的严重病害。课题组前期研 究表明具有P-loop 结构域的GmPR10（Gene Bank accession no. FJ960440)和具有P-loop、Bet v1 结构域的Gly m 4l （Gene Bank accession no. HQ913577.1)抑制大豆疫霉菌生长,并且过表达GmPR10 和Gly m 4l 的转基因大豆植株可 以提高对大豆疫霉根腐病的抗性。为研究GmPR10 和Gly m 4l 抑菌机理,本研究利用点突变技术,获得了GmPR10 的P-loop结构域突变体（Gly48/Thr48和Gly51/Arg51）、Gly m 4l的P-loop结构域突变体（Gly49/Ile49和Lys55/Pro55）、GmPR10 和Gly m 4l的P-loop结构域以及Gly m 4l的Bet v1结构域缺失突变体,并纯化回收相应突变体蛋白,进行体外抑制 大豆疫霉菌试验。结果表明,突变或缺失P-loop,Bet v1结构域的GmPR10和Gly m 4l失去了抑制大豆疫霉菌（Race 1）生长的能力,说明P-loop、Bet v 1结构域对GmPR10和Gly m 4l行使抑菌功能至关重要。     

水稻精细胞优势表达基因RSG6启动子的克隆及表达

利用已知的[i]RSG6[/i]基因序列和GenBank中提供的[i]RSG6[/i]前导序列设计引物,通过基因组DNA的PCR扩增得到长度为1 408 bp和1 173 bp的两个[i]RSG6[/i]启动子片段PB、PS,测定序列分析发现,PB、PS分别位于水稻第10和第4染色体上,PB、PS序列之间具有较高的同源性,且都含有大量的启动子元件。通过设计带有酶切位点的引物扩增出PB和PS的各两条缺失片段PB1、PB2、PS1、PS2,与质粒pBI221连接后得到中间载体pBI221 PB1、pBI221 PB2、pBI221 PS1、pBI221 PS2,再与质粒pBIN19连接构建出双元表达载体pBIN GUSB1、pBIN GUSB2、pBIN GUSS1、pBIN GUSS2,转化农杆菌EHA105,感染烟草叶片和烟草花粉,瞬时表达显示缺失片段PB1、PB2、PS1、PS2均具有启动子功能,且PB1、PS1的启动效率较PB2、PS2高。     

FocVel2基因对黄瓜枯萎病菌丝生长及毒力的影响

 由尖孢镰孢菌黄瓜专化型引起的黄瓜枯萎病是世界黄瓜生产上的一种毁灭性病害。本研究鉴定了尖孢镰孢菌黄瓜专化型丝绒蛋白的一个同源基因FocVel2,利用基因敲除和互补的方法研究该基因的功能。敲除突变体菌株ΔFocVel2出现明显的表型变化,包括菌落生长速率降低和产孢量降低,并且敲除突变株对黄瓜幼苗毒力明显减弱,回补突变体菌株能够恢复敲除突变体ΔFocVel2的所有缺陷。总之,研究结果发现丝绒蛋白基因FocVel2在菌体无性繁殖以及侵染过程中起到重要作用。     

SREBP1基因多态性与西门塔尔牛肌内脂肪含量相关性分析

选取165头西门塔尔育肥牛为研究对象,利用PCR方法检测固醇调节元件结合蛋白（Sterol regulatory ele-ment binding factor 1,SREBP1）基因第5内含子内84bp插入缺失突变的多态性,并与肌内脂肪含量进行相关性分析。结果显示,SREBP1基因第5内含内84bp插入缺失突变与棕榈油酸（C16：1）、硬脂酸（C18：0）、SFA和甘油三酯及C16脂肪酸不饱和指数显著相关（P〈0.05）,LL型个体棕榈油酸、甘油三酯和16碳脂肪酸不饱和指数均高于LS型个体,而硬脂酸和SFA低于LS个体。     

In vitro and in vivo efficacy of a live attenuated Salmonella Typhimurium vaccine at preventing intestinal colonization in chicks

Vaccination of chicks with Salmonella (S .) Typhimurium aroA deletion mutants has previously been shown to inhibit intestinal colonization of wild‐type S.  Typhimurium strains. In Australia, Bioproperties VaxSafe? STM1 strain is the only licensed and commercially available S . Typhimurium vaccine. This vaccine is a live attenuated aroA deletion mutant. Currently, it is recommended that the first dose of the STM1 vaccine is administered through coarse spray. It is unclear whether this mode of administration effectively permits intestinal colonization. Furthermore, it is not known whether the STM1 strain prevents or inhibits Salmonella colonization of chicks following this first dose. This study investigated both in vitro and in vivo colonization parameters. Invasiveness was assessed using an in vitro invasion assay into sections of ileum and caecum collected from day‐old chicks. The S.  Typhimurium definitive types (DT) 9 and 44 exhibited the greatest invasion into both intestinal segments. STM1 was invasive but was significantly less so than both isolates of S.  Typhimurium. In dual and triple infections, no competitive microbial interactions between STM1 and wild‐type Salmonella were observed. In vivo colonization inhibition was also tested. Vaccinated and nonvaccinated day‐old chicks were challenged with S.  Typhimurium DT9. Both STM1 and S.  Typhimurium DT9 were found in spleen, liver, ileum, caecum and caecal contents from day 2 postinfection. No significant exclusion effect was observed in vaccinated and challenged chicks.     

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。     

Genetic and molecular organization of the short arm and pericentromeric region of tomato chromsome 6

We have previously presented an integrated linkage map of tomato chromosome 6, that showed the position of restriction fragment length polymorphism (RFLP) and randomly amplified polymorphic DNA (RAPD) markers relative to a variety of classical markers. As for the short arm, map resolution has now been improved by crossing the chromosome 6 substitution line WSL6 to additional tester lines, carrying markers on the short arm. Molecular linkage analysis of the F₂ populations enabled us to produce an integrated linkage map showing the position of molecular markers relative to the classical markers Aps-1, yv, Mi, Cf-2/Cf-5, tl and pds. In order to incorporate the centromere into the integrated map, a radiation-induced deletion mapping strategy was applied, using irradiated pollen from L. pennellii LA716 in crosses to a L. esculentum line recessive for the markers yv and tl, that flank the centromere. Molecular analysis of the hemizygous yv-deletion and tl-deletion plants identified among the F₁ progeny, provided an estimate of the size of the respective deletions and, thus, of the position of the centromere relative to the molecular markers linked to yv and tl. This radiation mapping approach also provided evidence showing that, unlike published data, the root knot nematode resistance gene Mi as well as the Cladosporium fulvum resistance genes Cf-2/Cf-5 are located on the short arm.     

β-catenin基因26 bp插入/缺失对陕北白绒山羊生长性状的遗传效应分析

旨在研究β-连环蛋白(CTNNB1)基因上的多态性位点,并与陕北白绒山羊生长性状进行关联分析,寻找与生长性状相关的遗传标记,为山羊高生产力的标记辅助选择提供科学依据。以陕北白绒山羊(n=848)为研究对象,PCR大样本扩增检测β-catenin基因的插入/缺失(InDel)突变,并评估该位点与生长性状的相关性。结果发现,在848只陕北白绒山羊中β-catenin基因的第10内含子上存在一个26bp InDel的位点;该群体中共出现3种基因型,分别为II、ID和DD。关联性分析结果表明,该突变位点与陕北白绒山羊胸深显著相关(P<0.05),其中杂合型ID为优势基因型。因此,β-catenin基因可作为陕北白绒山羊生长性状选育的候选基因,为陕北白绒山羊选育工作提供理论依据。     

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.     

利用大豆鲜子叶快速检测脂氧酶缺失

脂氧酶是大豆产生豆腥味的主要原因,培育脂氧酶完全缺失大豆新品种可以从加工源头上解决加工去腥难题。在F2分离世代筛选脂氧酶缺失体是无腥味大豆育种的关键。本研究对现有的ISDS-PAGE电泳技术筛选方法进行改进,在苗后子叶展开期,取鲜子叶20 mg,快速无损测定脂肪氧化酶,由过去的播前切粉取样,改为苗后鲜子叶取样,改良后的方法不仅能清晰鉴别Lox-1,2,3三种同工酶缺失与否,同时确保含目标性状个体的正常生长发育,为大豆脂氧酶缺失育种提供技术支持。