苯并咪唑类抗性基因Benr在角毛壳菌中的转化

为提高角毛壳菌对苯并咪唑类杀菌剂的抗性,利用聚乙二醇介导的原生质体转化方法,以pBT6质粒为载体,把苯并咪唑类抗性基因Benr转化到角毛壳菌原生质体中,转化率约为1.5×10-5.通过Southern印记杂交分析,证明得到的转化子是阳性转化子.转化子对植物病原菌立枯丝核菌、杨树叶枯病菌有较强的抑制效果,能在多菌灵质量浓度为60 μg/mL的马铃薯葡萄糖琼脂培养基上生长.其抗药性比野生角毛壳菌提高约100倍,并且抗药性能够稳定遗传.研究获得了具有苯并咪唑类杀菌剂抗药性的角毛壳菌转化子.     

两种毛壳菌对几种植物病原菌的生防效果分析

将角毛壳菌和球毛壳菌与立枯丝核菌等5种植物病原菌进行对峙试验，结果表明，球毛壳菌和角毛壳菌对几种病原菌的抑制作用明显．球毛壳菌主要通过竞争作用、重寄生作用抑制病原菌，角毛壳菌主要通过产生红色拮抗物质、竞争作用抑制病原菌的生长．     

角毛壳菌丝切蛋白基因的克隆和特性分析

在构建的角毛壳菌(Chaetomium cupreum)菌丝体cDNA文库中获得丝切蛋白(Cofilin)的ESTs序列片段,为研究角毛壳菌的生物特性,提高其生物防治效果,以角毛壳菌总RNA反转录产物为模板进行PCR扩增,成功获得了Cofilin基因cDNA序列.生物信息学分析结果表明,该基因全长468bp,编码155个氨基酸,编码蛋白理论分子量17kD,等电点是4.99,α-螺旋和无规则卷曲是其主要的二级结构,为亲水性蛋白质.该蛋白有6个位点发生Ser磷酸化,4个位点发生Tyr磷酸化,有4个保守区,ClustalX分析说明该蛋白与柄孢霉(Podospora anserina)和球毛壳菌(C.g lobosum)亲缘关系较近.     

小球藻基因工程选择标记研究

研究了小球藻对10种常见抗生素:庆大霉素、新霉素、氨苄青霉素、卡那霉素、头孢霉素、链霉素、G418、潮霉素、Zeocin和氯霉素的敏感性,发现小球藻对庆大霉素、新霉素、氨苄青霉素、卡那霉素、头孢霉素和链霉素不敏感;对G418和潮霉素较敏感;对Zeocin和氯霉素最敏感.利用统计学的方法和原理,确定了G418、潮霉素、Zeocin和氯霉素在海水培养基和淡化10倍的培养基中对小球藻的半致死剂量和95%可信限.为筛选出小球藻基因工程藻株的选择试剂,建立其遗传转化系统奠定了基础.     

笋玉米原生质体培养及遗传转化的研究

以自花受粉的笋玉米幼胚为外植体建立了胚性细胞悬浮系,酶解游离原生质体通过液体浅层培养得到了再生植株;通过PEG法和电激法对原生质体进行了遗传转化,转化用外源基因是潮霉素抗性基因和BT基因,原生质体再生愈伤组织经潮霉素筛选后,PEG法和电激法获得抗性愈伤组织的频率分别为9 3%和8.9%,Southem-blotting证明外源基因已整合到玉米抗性愈伤组织细胞基因组中,转基因工程植株的分化工作在正在进行中.     

中国毛壳菌科分类研究 (Ⅱ)毛壳菌属一新记录种

从植物残体上和土壤中分离到一种毛壳菌,经鉴定为四角毛壳(Chaetomium quadrangulatum ).该菌主要鉴别特征为子囊果椭圆形或坛状;果壁细胞褐色,具棱角,花瓣状排列;顶生附属丝螺旋状卷;子囊棍棒状,具柄;子囊孢子四角形.该种为我国新记录种.     

法夫酵母整合型表达载体的构建

为了建立法夫酵母虾青素代谢途径研究和分子育种的技术体系,构建了法夫酵母的整合型表达载体.通过测定法夫酵母对G418的抗性来确定抗性筛选物的质量浓度,以来源于法夫酵母本身的rRNA基因为基因同源重组片段,利用法夫酵母肌动蛋白启动子和甘油醛-3-磷酸脱氢酶(gpd)启动子,构建了携带G418抗性基因的整合型载体pMD-18spakta和pMD-18spgktg.结果表明:法夫酵母对20μg/mL质量浓度的G418敏感,将构建的载体转化法夫酵母菌株后,筛选得到了能够在含有G418的培养基上生长的转化子;利用PCR的方法检测到转化子中存在的抗性基因.将筛选得到的阳性菌株连续传代培养10次后,发现该菌株的G418抗性仍然存在;以总DNA为模板,用PCR的方法仍可检测到G418抗性基因.说明已经构建得到了遗传稳定性良好的法夫酵母整合型表达载体,构建的质粒pMD-18spakta和pMD-18spgktg可作为法夫酵母整合载体应用于法夫酵母的DNA重组实验.     

毛壳菌产木聚糖酶条件的研究

研究了以玉米芯为原料制备粗木聚糖的最优生产条件:60℃浸泡1 h,冷藏8-10 h,乙醇洗涤用量为原木聚糖溶液的2-3倍。用三种球毛壳菌及两种中国新录毛壳菌对以玉米芯为基质的木聚糖酶产酶效果作对比实验,结果发现:球毛壳菌ACCCC30566产生的木聚糖酶具有较高的酶活性。     

BYDV CP基因转化小麦原生质体及转基因植株再生

用原生质体融合技术与ＰＥＧ介导的直接转基因方法相结合，使高频率分裂的小麦济南１７７悬浮细胞系原生质体与具有分化能力的小麦济南１７７胚性意伤组织原生质体融合，形成具有分裂和分化能力的融合细胞；同时，利用ＰＥＧ对原生质体的直接转基因作用，将带有抗病毒基因（ＢＹＤＶＣＹｇｅｎｅ）的质粒Ｐｐｐ１５与带有抗性选择标记基因的质粒严ＰＢｌＡｃｔＳＮ导入融合细胞中．经抗性筛选获得抗潮霉素（Ｈｍ）的阳性克隆并再生植株．对再生植株作ＰＣＲ检测表明，ＣＰ基因已整合到转化植株的基因组中并稳定表达．     

球毛壳菌引起杭白菊叶枯病的首次报道

对浙江磐安地区发生叶枯症状的杭白菊进行病原菌分离鉴定,经形态学和分子生物学分析发现病原菌为球毛壳菌（Chaetomium globosum）。致病性测定后显示该菌能引起杭白菊枯叶和叶边缘萎缩等症状,与田间观察到的症状一致,符合科赫氏法则,这是目前国内外首次报道的一种新病害。     

Chaetoomium globosum与Panus rudis共培养发酵合成漆酶

研究Chaetoomium globosum与Panus rudis在共培养条件下的漆酶合成.平板对峙培养时,菌丝体生长呈僵持状态,接触区漆酶表达量提高;摇瓶发酵实验表明,C.globosum与P.rudis共培养时,漆酶活力达4 360 U.L-1(ABTS法),是P.rudis单独培养的14倍,而C.globosum单独培养时无漆酶分泌;改变初始发酵时添加的C.globosum菌丝体量,或者延长发酵时间,漆酶同工酶谱发生改变.     

桉树抗桉树枝瘿姬小蜂理化因子的多元回归分析

【目的】通过对桉树抗桉树枝瘿姬小蜂(Leptocybe invasa)相关的多个理化指标进行多元回归分析,确定与抗虫性最紧密关联的关键因子,为筛选抗虫桉树品系提供理论指导。【方法】首先比较了窿缘桉Eucalyptus exserta(EA)、巨圆桉Eucalyptus grandis×Eucalyptus tereticornis(DH201-2)和巨尾桉Eucalyptus grandis×Eucalyptus urophylla(G9)3个桉树品系对桉树枝瘿姬小蜂的抗性强弱,同时测定了桉树品系间叶片含水率、叶片厚度、比叶面积(Specific leaf area,SLA)、叶片C含量、N含量、C/N比、总酚、缩合单宁等8个理化指标,最后通过多元逐步回归分析筛选出与抗虫性紧密关联的理化因子,并建立回归方程。【结果】桉树枝瘿姬小蜂不能在G9上产生虫瘿,在DH201-2上的样枝平均虫瘿数显著高于EA。3个桉树品系叶片厚度间无显著差异;DH201-2叶片含水率显著高于EA和G9;N含量表现为DH201-2EAG9;G9叶片C含量、总酚和缩合单宁含量显著高于EA和DH201-2。通过多元逐步回归分析,最终进入模型的是缩合单宁(X_2)和C含量(X_3);建立了以抗性得分为因变量(Y),缩合单宁含量(X_2)、C含量(X_3)为自变量的回归方程:Y=-20.671+2.095X_2+0.433X_3(复相关系数R=0.770,校正相关指数R~2=0.578,显著水平P0.001)。【结论】缩合单宁和C含量是影响桉树对桉树枝瘿姬小蜂抗性强弱的关键因子,且两个因子与抗虫性正相关;所建立的回归方程可以很好地预测桉树抗虫性与两个化学指标间的相关关系,为今后选育桉树抗虫优良品系提供理论依据。     

Genetic defect in secretion of complement C5 in mice.

A genetic deficiency of the fifth (C5) component of complement1-3, a serum glycoprotein of molecular weight (MW) 220,000 (ref. 4), has been found in 39% of inbred strains of mice3. Sera of deficient mice lack detectable C5 activity and protein2,3. In addition deficient mice produce antibody to mouse C5 when injected with sera from C5 sufficient (normal) strains. Levy et al.5 showed that somatic cell hybrids between C5 deficient (B10.D2/old line) macrophages and either C5 sufficient (B10.D2/new line) mouse kidney or chicken erythroblasts secreted haemolytically active mouse C5 in vitro. Several possible molecular mechanisms to account for the findings were considered, but insufficient direct data were available to choose among them. We recently reported that mouse (CD.1 strain) peritoneal cells in culture synthesise and secrete a single chain precursor, pro-C5 (MW approximately 210,000), of the two-chain (alpha chain, 125,000 and beta chain 83,000 MW) C5 protein6. Radiolabelled precursor C5 was contained within the cells and was secreted into the tissue culture media. Using similar methods, we now find that C5 deficiency in each of five different mouse strains (AKR, SWR, DBA/2J8 A/HeJ and B10.D2/old line) is due to a failure in secretion of C5 protein and not to a failure in biosynthesis of pro-C5.     

High expression of EPO gene using un-dhfr negative cell

Erythropoietin (EPO) genomic gene was cloned and its expression vector pOP13/EPO was constructed. CHO_K12 cell was transfected by this vector using lipofectin method. A stable expression cell strain C10 cell with the EPO production at 160IU/d in 10\+6 cells were obtained at 400 μg/mL G418. Based on the C10 cell, another vector pHY/dhfr (dihydrofolate reductase) that carries a dhfr gene and a selecting marker of hygromycin B resistant gene was transferred to this cell. Several cell clones were obtained at 200 μg/mL hygromycin B. These cell clones that can express both EPO gene and exogenous dhfr gene were selected under the progressively increased concentration to 1 μmol methotrexate(MTX). Some high EPO expression cell clones were obtained, the highest expression was 2 400 IU/d in 10\+6 cells, 15 times higher than that without MTX pressure. Then, a method of EPO high expression by using un_dhfr negative cell was primarily established. EPO bioactivity was found by using TF_1 cell.     

Construction and expression of inverted configuration of retroviral vector containing intron 1 of hFIX

Intron was found to play an important role in improving gene expression. To improve the human factor IX(hFIX) expression level in hemophilia B gene therapy study, the retroviral vector containing intron 1 of hFIX gene was constructed in forwarded configuration, but the intron 1 was found spliced in virus particles by RT_PCR detection. So the inverted configuration vector G1NaPAi′IX was suggested and constructed on the basis of SNMBAIXm and transfected into PA317. Then C2C12 cells were transfected using the above virus supernatant and the G418_resistant clones were selected. PCR and RT_PCR detection found that intron 1 structure existed in C2C12 clones and retroviral particles. And the expression level of inverted vector was 3 times higher than that of forwarded vector. These results showed that the inverted configuration vector was in deed able to avoid splicing of intron 1 during the process of retroviral packaging and improved the expression level of hFIX protein.     

Expression of a transposable antibiotic resistance element in Saccharomyces

Some eukaryotic genes can be expressed in bacteria but there are few examples of the expression of prokaryotic genes in eukaryotes. Antibiotic G418 is a 2-deoxystreptamine antibiotic that is structurally related to gentamicin but has inhibitory activity against a much wider variety of pro- and eukaryotic organisms. In bacteria, resistance to G418 can be determined by several plasmid-encoded modifiying enzymes and, in view of the broad spectrum of activity of G418, we considered that this antibiotic might be useful as a selective agent for the introduction of these antibiotic resistance genes into a eukaryotic organism such as Saccharomyces cerevisiae. Additional impetus for these experiments came from the knowledge that certain of the G418-resistance determinants in bacteria are carried on transposable elements; a study of the properties of these elements in eukaryotes would be intriguing.     

抗生素在体内外对肠道厌氧菌的作用

在体外实验中,自肠道分离的95株厌氧菌菌株对氯林霉素、甲砜霉素和灭滴灵都比较敏感。用氯林霉素[2.5 mg/(d·小鼠)]处理只结合有厌氧菌并能拮抗艰难梭菌肠道定居的悉生小鼠,可引起菌群屏障可逆性的破坏,使艰难梭菌接种物能在肠道中定居繁殖;在艰难梭菌菌数达到一定水平时,可在动物盲肠中检测到艰难梭菌细胞毒素。文中根据实验结果讨论了与抗生素疗法有关的伪膜性结肠炎产生的可能机制。     

栽培棉(4X)与斯特提棉(2X)种间杂种F_1的细胞学的研究

本研究分析了陆地棉[Gossypium hirsrtum L.,2n=4x=52,(AD)1]×斯特提棉[G.sturtianum willis,2n=2x=26,C_1]和海岛棉[G.barbadense L.,2n=4x=52,(AD2)]×斯特提棉二个种间杂种F_1花粉母细胞减数分裂时的染色体行为,并观测了杂种F_1的花粉粒大小和生活力。根据各杂交组合染色体配对表现,讨论了这些棉种间的亲缘关系和棉花种间杂种的利用问题。     

Cytogenetics of intergeneric hybrids between Brassica species and Orychophragmus violaceus

In the sexual intergeneric hybrids between the cultivated Brassica species and Orychophragmus violaceus, both complete separation and partial separation of the parental genomes were found to occur during mitosis and meiosis under genetic control. The cytogenetics of these hybrids was species-specific for Brassica parents. The different chromosome behavior of hybrids with three Brassica diploids ( B. campestris , B. nigra and B. oleracea ) might contribute to the different cytogenetics of hybrids with three tetraploids ( B. napus, B. juncea and B. carinata). Owing to the parental genome separation, Brassica homozygous plants and aneuploids with various chromosome constitutions were identifiable in the progenies of these hybrids, which were valuable for the study of the structure and evolution of Brassica genome and for the breeding of Brassica crops.