趋磁螺菌AMB-l磁小体合成相关基因的克隆及功能分析

为了更清楚的了解趋磁螺菌产磁小体的合成机理和调节途径,用Tn5转座子诱变的方法筛选得到了2株磁小体合成降低的突变株,并克隆了突变株中被插入失活的基因,分别为编码ABC型Fe3+转移系统中的离子结合蛋白的amb3385基因和功能未知的amb3672基因.互补实验表明携带amb3385和amb3672基因的广宿主载体可以不同程度地恢复突变株中磁小体的合成,证明了D.Schüler关于磁小体合成假说的第一个步骤,即Fe3+从胞外向经由Fe3+转运蛋白运输至了胞内.由于amb3672基因比对时未发现特殊相似基因,其功能尚需进一步研究.     

Screening of the interacting proteins with NifA in <Emphasis Type="Italic">Azospirillum brasilense</Emphasis> Sp7 by the yeast two-hybrid system

NifA in AzospiriUum brasilense plays a key role in regulating the synthesis and activity of nitrogenase in response to ammonia and oxygen available. In this work we used the yeast two-hybrid system to identify the proteins that interact with NifA. The nifA gene was fused to the yeast two-hybrid vector pGBD-C2, and three A. brasilense Sp7 genomic libraries for use in yeast two-hybrid studies were constructed. Screening of the libraries identified four clones encoding proteins that interact with NifA. The confirmation of the interactions of each gene product of the four clones and NifA were carried out by exchanging the vectors for nifA and the four clones and by mutageneses of the four clones with shift reading frame experiments in yeast two-hybrid studies. DNA sequence analyses showed that two clones encode proteins containing PAS domains that play an important role in signal transduction. One clone has high similarity with the fhuE gene of Escherichia coli, whose gene product is involved in iron uptake and transportation, and the other clone encodes an unknown protein.     

甘露醇和AgNO_3对小麦细胞再分化的影响

比较了甘露醇和AgNO3 对小麦 (TriticumaestivumL.)品种保7059幼穗细胞再分化和IAA氧化酶、IAA过氧化物酶活性的影响 .结果表明 ,AgNO3 能够明显提高小麦愈伤组织再生率 ,但甘露醇使其再生率降低 ,AgNO3 对IAA氧化酶和IAA过氧化物酶活性无明显影响,但甘露醇使2种酶活性明显升高 .     

Isolation and characterization of a <Emphasis Type="Italic">Sinorhizobium fredii</Emphasis> mutant that cannot utilize proline as the sole carbon and nitrogen source

Sinorhizobium fredii strain HN01 can use proline as the sole carbon and nitrogen source. A mutant strain GXHN100 unable to catabolize proline was screened from 6000 Tn5gusA5 random insertional mutants of S.fredii strain HN01. Sequencing analysis showed that an open reading frame, named pmrA (proline metabolic relative), was inserted by the Tn5gusA5. A positive clone, namedp GXHN100 which containing 3.3kb foreign DNA fragment of S.fredii strain HN01, was isolated from a partial gene library of S.fredii HN01 by colony in situ hybridization. Sequence analysis showed that pGXHN100 contained the entire pmrA gene. The 3.3kb DNA fragment of pGXHN100 was cloned into a broad-host-range cosmid vector pLAFR3 to form plasmid pGXHN200 which was subsequently introduced into GXHN100 to form a complemented strain GXHN200. Plant test showed that GXHN100 was effective and no obvious changes in nitrogenase activity comparing with parental strain. But GXHN100 nodulated 2 days later on soybean and its nodulation efficiency and competitiveness were decreased.The complemented strain GXHN200 restored the nodulation efficiency and competitiveness of GXHN100 to the wild type.     

甘蓝下胚轴不同切段内源IAA水平与不定根发生能力的研究

对甘蓝下胚轴不同切段的不定根发生能力的研究结果表明,从切段1到切段5都不需要任何激素的诱导,能“自发生根”,且不定根发生能力逐步增强。采用酶联免疫法（ELISA）测定了不同切段的内源IAA含量,进一步又对不同切段的过氧化物酶和IAA氧化酶相对活性以及不定根发生的组织学进行了研究,结果表明,内源IAA水平和不定根发生能力不呈正相关,下胚轴不同切段的根原基原始细胞可能处在不定根发端早期的不同时期。作者认为,在甘蓝下胚轴不同切段”自发生根系统“中,内源IAA水平和不定根发生能力不呈正相关,可能是由于该切段的根原基原始细胞已越过了它们分化不定根所需的IAA峰。     

补充照光对番茄幼苗生长和结果的影响

研究了补充照光对大棚番茄幼苗生长及其叶片IAA合成、降解酶活性的影响.结果表明,补充照光可使番茄株高明显降低,使其单株干重明显增高,但对其单株鲜重及茎粗无明显影响.补充照光还使番茄叶片叶绿素a,叶绿素b,叶绿素a/叶绿素b比值和叶绿素/类胡萝卜素比值明显降低,但引起类胡萝卜素含量极显著增加.补充照光不仅能使番茄果实提前成熟,还在较大程度上提高了番茄产量.补充照光使番茄幼苗叶片醛氧化酶、过氧化物酶和IAA过氧化物酶活性显著下降,但使IAA氧化酶活性明显增高.     

茄假单胞菌寄主花生特异性毒性基因的定位

通过对从茄假单胞菌中克隆的pGX1252进行亚克隆分析,发现含pGX1252右边4.5kbKpnI/EcoRI片段的亚克隆pGX1404仍象pGX1252一样能使对花生不致病菌株T2014在花生上致病.用转座子Tn5-loc对pGX1404进行了饱和插入诱变,一共分离得到6个在pGX1404上不同位置的独立插入突变,其中离pGX1404右末端约1.4kb、2.2kg的两个Tn5-loc插入使pGX1404丧失了扩大T2014寄主范围的能力,证实hsv基因位于pGX1404的右边.     

观赏羽扇豆生根过程中内源IAA和ABA含量变化

采用酶联免疫方法（Enzyme linked immunosorbent assay,ELISA）,对观赏羽扇豆品种尖塔（Minaretie）和奖品（Russell Prize）组培苗生根过程中内源吲哚乙酸（3-Indolyl-acetic acid,IAA）和脱落酸（Abscisic acid,ABA）含量进行了测定,总结IAA和ABA含量与组培生根的相关性,为其微体快繁和工厂化生产提供理论依据。结果表明：（1）IAA含量变化为单峰曲线,呈现“升高-降低-升高”的变化趋势;（2）ABA含量在不定根形成期呈下降并达到最低;（3）IAA含量与生根指数正相关,ABA含量与生根指数负相关,二者协同影响生根。     

Regulation of eight avr genes by hrpG and hrpX in Xanthomonas campestris pv. campestris and their role in pathogenicity

Eight putative avirulence genes in Xanthomonas campestris pv. campestris (Xcc) strain 8004 were characterized by Tn5gusA5 mutagenesis and gene expression analysis. The virulence test of mutants on Chinese radish showed that all mutants in individual avr genes except avrBs2 mutant were not significantly different from the wild type in virulence. The avrBs2 mutant showed reduced virulence and bacterial growth in planta. Gene expression analysis using β-glucuronidase as reporter indicated that avrBs1.1，avrBs1，avrXccB，avrXccC，avrXccE1 were regulated by hrpG, whereas avrXccA1, avrXccA2 and avrBs2 were not. RT-PCR analysis showed that all hrpG-regulated genes except avrBs1 were also regulated by hrpX. In addition, it was demonstrated that avrBs1　 was responsible for elicitation of a type III dependent hypersensitive reaction (HR) on nonhost plant pepper ECW-10R, and wild type Xcc 8004 was unable to cause HR on pepper ECW-20R.     

Tn5转座子插入p95基因不影响AcMNPV的复制

从Tn5转座子介导的AcMNPV随机插入突变体库中,分离到一株复制正常的突变体AcApra41.突变定位发现Tn5转座子插入了病毒p95基因中.为了排除AcApra41中还有其他突变,利用同源重组法构建了p95基因定点插入突变的重组病毒AcGFP-P95in.PCR确认p95基因中插入了Tn5转座子;Western blot也证实AcApra41和AcGFP-P95in感染的细胞中,P95蛋白的分子量都因为插入突变而变小,由野生型的95 ku变为 55 ku.病毒复制动态曲线和荧光显微镜观察证实带有该插入突变的病毒能够在Sf9细胞中正常复制,并表达极晚期基因.这一结果表明完整的P95蛋白对病毒复制是非必须的.     

The secretion of lecithinase of Pseudomonas alcaligenes S2 was via type II secretion pathway

Phosphorus is one of the major essential macronutrients for virtually metabolic processes in plant growth and de-velopment[1]. This creates a paradox with major agro-nomic implications since the phosphate form of phospho-rus is one of the least soluble mineral nutrient ions in the soil. The concentration of soluble phosphorus in soil is usually very low, normally at levels of 1 ppm or less (10 mol/L H2PO4?). Mineral forms of phosphorus are repre-sented in soil by primary minerals, such as ap…     

云南松苗期生长对激素浸种的响应

 云南松苗期生长极其缓慢,尤其是造林3a内具有严重的蹲苗现象.为此,研究试图通过IAA,IBA浸种对其实施促成培育.试验采用3×3回归设计并在田间排列上汲取拉丁方和随机排列的特点进行优化布设,从而建立了最优激素效应方程,据此探讨了激素效应规律、确定了激素最佳用量及最佳配比.结果表明:① 生长量及生物量与激素用量之间的关系呈钟形曲面模式,即各生长指标均存在一个产量峰值.峰值以前,各生长指标随激素用量的增大而提高;峰值以后,各生长指标随激素用量的增大而下降.② 不同生长指标对激素用量及其配比的响应规律具有一定差异,IAA,IBA配合浸种对生物量积累的促进作用依次为树干、叶片和根系,而较高比例的IBA有利于树干的高生长和根系发育、更高比例的IBA有利于树干的粗生长以及树干和叶片的生物量积累.③ 根据激素效应方程,求得最大时的最佳激素用量及最佳配比.其中,最佳IAA用量分别为50,57,53,49,45mg/kg,最佳IBA用量分别为86,143,88,99,107mg/kg,最佳IAA:IBA配比质量分别为1:1.72,1:2.51,1:1.67,1:2.03和1:2.37,对应的苗高、地径生长量及根、干和叶片生物量理论最高产量分别为8.00cm,0.51cm,0.380g,0.387g,1.230g,分别较对照提高了22.64%,66.12%,62.40%,198.00%,83.30%.     

Transmembrane structure and function of dctPQM encoding C4－dicarboxylate transport proteins from nitrogen－fixing P. stutzeri A1501

C4-dicarboxylate transport proteins of diazotroph Pseudomonas stutzeri were encoded by dctPQM genes. Nucleotide sequence analysis indicated that dctP, dctQ,and dctM grouped together. Its nucleotide and amino acid sequence shared high homology with that of dctP gene encoding periplasmic C4-dicarboxylate-binding protein and dctQM genes encoding C4-dicarboxylate transport proteins from the free-living nitrogen-fixing Aotobacter vinelandii.Structural analysis showed that DctP of P. stutzeri did not include membrane-spanning regions, and DctQ and DctM contained 5 and 12 transmembrane segments, respectively.The fragment containing the complete dctPQM genes was cloned into the Tn5 transposon region of suicide mobilization plasmid pSZ2L The resultant plasmid was named pSZY6.By triparental mating, Tn5 transposon carrying the dctPQM genes inserted into the genome of the wild type strain A1501,randomly. The recombinant strain A-142 which harboured an extra copy of dctPQM genes was constructed and identified by PCR amplification of npt II gene. When A-142 was grown in minimal medium with different concentrations (20,10 and 5mmol/L) of C4-dicarboxylates succinate, malate, or fumarate as the sole carbon source, the rate of nitrogen fixation assayed by acetylene reduction was significantly higher than that of the wild-type strain A150L This result was established that an extra copy of dctPQM genes could increase the activity of nitrogen fixation of P. stutzeri strain A1501.     

影响分光光度法测定IAA含量因素的研究

测定了叶绿素、高岭土、活性炭、不同加热时间、蛋白质、不同pH等条件对分光光度法测定IAA含量的影响。结果表明 ,叶绿素对IAA含量的测定有影响 ,而且随浓度的增大 ,其影响也增大 ;活性炭对IAA含量的测定有极显著的影响 ,在吸附色素的同时也吸附了IAA ;高岭土对IAA含量的测定没有影响。煮沸时间对测定结果有影响 ,短时间 (5min)煮沸对测定结果影响不大。样品中蛋白质的含量对测定结果有极显著的影响。体积分数为 0 10的三氯乙酸对测定结果影响不显著     

杉木根际固氮菌筛选及其溶磷性与分泌IAA特性研究

我国特有速生用材林主要造林树种杉木的人工林地衰退问题日益严重,但对杉木甚至木本植物根际促生菌的作用及其分离与利用等研究还很少报道.利用Ashby培养基和固氮酶乙炔还原测定法从杉木根际土壤中分离到了16株固氮能力较强的菌株,并在蒙金娜培养基上鉴定了溶磷圈、用钼锑钪比色法测定了有机磷和无机磷的溶磷能力、用Salkowski比色法测定了IAA分泌量.结果表明:16株菌中有5株固氮酶活性大于150 nmol.mL-1.h-1,最高的NGJ-4达264.7 nmol.mL-1.h-1,NGX-5次之,为237.4 nmol.mL-1.h-1.8株解无机磷,其中NGJ-8、NGJ-4和NGX-3的有效磷增量分别为182.7、158.4和133.9mg.L-1,但都无明显解有机磷能力.有5株菌解有机磷,但能力都较小,最大的NGX-5有效磷增量也仅为46.1 mg.L-1,且不具有解无机磷能力.大部分菌株都能分泌生长素,有6株菌分泌能力较强,培养液上清中IAA浓度大于20 mg.L-1,其中NGX-5、NGJ-3、NGJ-4和NGJ-8分别为35.8、32.1、27.8和27.7 mg.L-1.综合这些结果,菌株NGJ-4、NGX-5和NGJ-8兼有固氮、解磷和分泌IAA等3种最强或较强能力,在多功能微生物菌肥研制中具有重要利用价值.     

人参体细胞胚胎发生过程中的生理变化

采用考马斯亮蓝G-250染色法、蒽酮法、愈创木酚法等方法,研究了人参体细胞胚胎发生中可溶性蛋白、多糖及淀粉含量和POD及PPo活性的变化,并对体细胞胚发生的不同时期培养物内源LAA和ABA含量进行了测定.结果表明,多糖和淀粉含量在早期胚时较低,可溶性蛋白含量、POD及PPO活性和IAA含量在早期胚时最高;在成熟胚时期,ABA含量最高,且ABA与IAA的比值在成熟胚时也较高.说明多糖和淀粉参与调节胚性细胞的分化和发育过程,POD及PPO和可溶性蛋白与早期胚形成密切相关;ABA与IAA比值高有利于体细胞胚的发育成熟.     

青钱柳种子综合处理过程中内源激素的动态变化

青钱柳种子属于深休眠类型,在自然条件下,需要经过隔年甚至2年才能萌发,笔者对综合处理的青钱柳种子萌发过程中各种激素的变化进行分析。结果表明:随着层积时间的增长,其脱落酸(ABA)的含量逐渐减少,而赤霉素(GA)、生长素(IAA)、细胞分裂素(CTK)的含量则逐渐增加。赤霉素与脱落酸、生长素与脱落酸和细胞分裂素与脱落酸含量的比值也呈现出基本增加的趋势。进一步证实了赤霉素(GA)是种子萌发的促进物质。同时,生长素(IAA)、细胞分裂素(CTK)在解除种子休眠过程中也起到一定的促进作用。     

植物生长调节物质对山核桃嫁接的效用

采用3种植物生长调节物质（IAA、6－BA、GA1，3）以3种浓度对山核桃3类接穗（一年生枝、生长枝、结果枝）的嫁接进行正交田间试验（L273^3)，经方差分析表明：试验的3种物质中，IAA具显作用，F值分别为2．91、2．94、3．49，都超过了F0．10临界值；6－BA没有效果，赤霉素在结果枝的嫁接中与生长素交互起显作用。因此，在山核桃嫁接时，外源IAA对山核桃嫁接起主导作用，不同时间的处理，激素对嫁接的效果不同。