苜蓿根瘤菌聚羟丁酸代谢突变体的竞争生长和结瘤能力以及外源生物素的影响

对苜蓿根瘤菌(Sinorhizobium meliloti)聚羟丁酸(PHB)代谢突变体与野生型菌株之间，以及不同突变体之间的竞争生长和竞争结瘤能力在不同培养条件下进行了测定，并研究了外源生物素对各突变体竞争生长和竞争结瘤能力的影响。结果表明：①phbC突变体菌株与野生型菌株共培养，不论培养基中添加、不添加外源生物素，phbC突变体均表现出生长竞争能力的严重缺陷；竞争结瘤实验也显示，该突变体同野生型菌株竞争结瘤能力大幅下降；说明PHB合成能力的缺陷影响了菌株的竞争生长和竞争结瘤能力。②bdhA突变体与野生型菌株共培养，在不添加外源生物素的情况下，bdhA突变体同野生型菌株竞争生长的能力有明显缺陷，但在添加外源生物素的情况下，其竞争生长能力有明显提高；bdhA::Tn5突变体与phbC::Tn5-233突变体共培养。如培养基中不添加外源生物素，二者间的竞争生长能力无大的差异；但若添加外源生物素，则bdhA突变体的竞争生长能力明显高于phbC突变体；表明外源生物素对bdhA突变体的竞争生长能力有重要作用。     

丝氨酸羟甲基转移酶基因(glyA)的克隆

采用PCR方法从大肠杆菌K12染色体DNA中克隆出1．5kb的DNA片段，并将其与pUCm—T载体连接转入大肠杆菌JMl09中。DNA测序分析证明该片段含有一个完整的丝氨酸羟甲基转移酶基因（glyA)。     

慢生型大豆根瘤菌菌株GX201的脯氨酸脱氢酶基因(putA)的分子克隆

根据已报道的脯氨酸脱氢酶基因（ｐｕｔＡ）序列,通过ＰＣＲ方法,从慢生型大豆根瘤菌菌株ＧＸ２０１的总ＤＮＡ中扩增到—ＰＣＲ产物。序列分析表明,该ＰＣＲ产物的长度为４１８ｂｐ,与已报道的ｐｕｔＡ基因具有９３．５％的同源性。以广谱寄主范围质粒ｐＬＡＦＲ３为载体,在大肠杆菌ＤＨ５α中构建了ＧＸ２０１的基因文库,并以该ＰＣＲ产物为探针,从基因文库中筛选到一重组质粒ｐＧＸＮ３００。     

浙粳22类病斑突变体spl(t)特征及其基因定位

在浙粳22辐照的突变体库中,筛选出一个对环境不敏感的全生育期表达的类病斑突变体。遗传分析表明,该突变性状由1对隐性核基因spl(t)控制。利用SSR标记对类病斑突变体spl(t)与珍汕97B杂交构建的F2群体进行基因定位,把spl(t)基因定位在第12号染色体短臂端的着丝粒附近,位于SSR标记RM7195与RM27929之间的0.8cM区间内。锥虫蓝染色检测结果表明spl(t)类病斑的形成和发展是一个程序性细胞死亡的过程。进一步研究表明,这种程序性细胞死亡是由H2O2氧迸发而致。突变体经白叶枯病和稻瘟病鉴定,表明该突变体的抗病性与野生型品种浙粳22相仿。     

根瘤菌中的nodD基因及其与弗兰克氏菌（Frankia）中的类nodD基因的功能互补

根瘤菌可在几种豆科植物上成瘤,结瘤基因（ｎｏｄ基因）赋予这些细菌以寄主专一性的方式诱导瘤的形成。目前,已被鉴定的三类主要结瘤基因是：＂公共＂结瘤基因,如ｎｏｄＡＢＣ,这类基因是结瘤不可缺少的,并具有高度的结构和功能上的保守性;另一类是寄主专一性基因,这类基因具有种属或菌株的专一性,它们决定了细菌的寄主范围;最后一类是结瘤基因Ｄ（ｎｏｄＤ）,这类基因编码一类调节蛋白,并和植物分泌的酚类化合物一道起动其他结瘤操纵子的转录。一些根瘤菌种属只有一个拷贝的ｎｏｄＤ基因,而另一些则可拥有三个拷贝之多。苜蓿根瘤菌（Ｒ．ｍｅｌｉｌｏｔｉ）中存在三个拷贝的ｎｏｄＤ基因,其中的任何一个发生突变都以依赖于寄主的方式影响结瘤效果。在慢生型大豆根瘤菌（Ｂ．ｊａｐｏｎｉｃｕｍ）中,ｎｏｄＤ１突变菌株还可在大豆或其他豆科植物寄主上结瘤,只是稍往后推迟而已,ｎｏｄＤ２基因对ｎｏｄＹＡＢＣＳＵＩＪ操纵子诱导的影响却很小,两个ｎｏｄＤ拷贝突变后尚表现出结瘤活性。在只有一个ｎｏｄＤ基因拷贝的根瘤菌种属中,如豌豆根瘤菌（Ｒ．ｌｅｇｕｍｉｎｏｓａｒｕｍ）和茎瘤固氮菌（Ａ．ｃａｕｌｉｎｏｄａｎｓ）中,ｎｏｄＤ突变后会导致菌株丧失结瘤能力。一般说来,不     

pBI121-Lyz-GFP表达载体的构建及其在和田苜蓿愈伤组织中的转化

将绿色荧光蛋白(GFP)基因片段插入到pBIl21-Lyz多克隆区,构建了重组表达载体pBI121-Lyz-GFPo经Sma Ⅰ与BamH Ⅰ双酶切和PCR验证重组质粒,均能得到约750 bp的目的片段,通过进一步测序证明,GFP基因已成功地连接到pBI121-Lyz中,且重组质粒连接方向正确.利用冻融法将重组质粒导人根癌农杆菌(Agrobactcrium tumenfaciens)LBA4404,用叶盘法转化和田苜蓿(Medicago sativa L.cv.Xinjiang Daye),筛选出具有卡那霉素抗性的愈伤组织,经PCR扩增和荧光显微镜检测证明,重组基因已成功地转化到和田苜蓿愈伤组织中.     

pBI121-LyrZ-GFP表达载体的构建及其在和田苜蓿愈伤组织中的转化

将绿色荧光蛋白（GFP）基因片段插入到pBI121-Lyz多克隆区,构建了重组表达载体pBIl21-Lyz-GFP。经SmaⅠ与BamHⅠ双酶切和PCR验证重组质粒,均能得到约750bp的目的片段,通过进一步测序证明,GFP基因已成功地连接到pBI121-Lyz中,且重组质粒连接方向正确。利用冻融法将重组质粒导入根癌农杆菌（Agrobacterium tumenfaciens ） LBA4404,,用叶盘法转化和田苜蓿（Medicago sativa L. cv. Xinjiang Daye）,筛选出具有卡那霉素抗性的愈伤组织,经PCR扩增和荧光显微镜检测证明,重组基因已成功地转化到和田苜蓿愈伤组织中。     

用荧光酶基因(cfp)标记法研究菜豆根瘤菌的竞争性和有效性

试验采用三亲本杂交法将cfp基因导入一株有效性较高的菜豆根瘤菌SC18,获得了一株带cfp基因标记的菌株SC18-cfp。对SC18-cfp进行性状、标记基因的遗传稳定性检测,结果表明,cfp基因不仅能有效表达,而且性状稳定。随机挑取SC18-cfp的10个转移结合子在无氮砂培条件下进行标记菌株与亲本菌株在3个菜豆品种上的竞争结瘤试验。结果证实,10个转移结合子在供试植物根系上的占瘤率均接近50%,其有效性和竞争结瘤能力与亲本菌株间的差异不显著,品种间的差异性不显著;在控制田间试验中SC18-cfp比土著根瘤菌的竞争结瘤能力强,在品种A、B、C上的占瘤率分别达到了57.8%、60.0%、55.6%。接种SC18的植株干重、全氮量、叶绿素含量和豆荚产量均显著高于对照。     

花生~(60)Co-γ辐射诱变和突变体库的构建

为了获得花生新种质,对花生干种子进行60Co-γ辐射诱变处理并构建花生突变体库,获得了一些有价值的突变体材料,如高油突变体、高蛋白突变体、大种子和小种子突变体、大叶和小叶突变体、卷叶突变体等。通过分析这些突变体的不同性状发现,辐射处理对株高、叶片形态、含油量、蛋白含量、单株结荚数、单粒重、油亚比、出仁率等性状的影响极为显著。对7个性状(单株果仁重、蛋白含量、亚油酸含量、油酸含量、含油量、单粒重和单株荚果重)进行相关性分析,结果表明,油酸含量与亚油酸含量为高度线性相关;含油量与蛋白含量、亚油酸含量、油酸含量均为显著性相关;而含油量与单株荚果产量呈低度线性相关。这些突变体的获得为今后进行花生遗传育种以及机理研究提供了可靠的原材料和中间材料,也为花生功能基因组学研究提供了丰富的材料。     

家蚕胞质肌动蛋白基因启动子的克隆及piggyBac转座子表达载体的构建

摘要: 利用PCR方法从家蚕（Bombyx mori）总DNA中克隆到细胞质肌动蛋白基因（cytoplasmic actin）启动子片段，序列分析表明，该片段中含有典型的组成型表达的细胞质肌动蛋白基因A3（BmA3）调控序列：SRE元件（serum response element）和ActE1元件(active element 1)。该序列的核苷酸序列与来自欧洲的一个家蚕品种的序列同源性为94%。通过多次重组，将A3启动子片段(不含信号肽序列)与转座子 piggyBac的转座酶编码区融合构建成辅助质粒；将其(含信号肽序列和部分编码区)与报告基因多管水母绿色荧光蛋白基因gfp融合，插入到人工合成的转座子piggyBac两反向重复末端序列之间，进而构建成基于转座子piggyBac的转基因载体。研究中构建的转座子转基因载体仅6.4 kb，并在两反向重复末端序列之间设计了1个多克隆位点区，便于目的基因的插入。     

Survival and persistence of genetically modified Sinorhizobium meliloti in soil

Studies were conducted to evaluate the survival and persistence of Sinorhizobium meliloti 104A14 and two acid phosphatase-negative mutants in Kirkland (fine, mixed, thermic Udertic Paleustolls) silt loam soils with various fertility levels, and to assess the impact of inoculation on nodule occupancy and soil microbial community structure in the inoculated alfalfa (Medicago sativa L.) rhizosphere. Recovery of the inoculated strains was 100% (in the order of 10⁸ cells g⁻¹ soil) immediately following inoculation to soils, but decreased from 10⁸ cells g⁻¹ soil to undetectable levels in a nutrient-poor soil within 32 days. In a nutrient-rich soil, approximately 2–3% (4.7–7.43×10⁶ cells g⁻¹ soil) of the mutants and 23% (5.84×10⁷ cells g⁻¹ soil) of the wild-type inocula persisted for more than 64 days. Survivability and persistence of the wild-type S. meliloti were significantly greater than that of the genetically modified acid phosphatase negative mutants in all the soils tested. The persistence and nodule occupancy of the introduced S. meliloti in sterile and non-sterile soils were also tested for two repeated alfalfa growth periods in the same plant growth units, with a 1 month interval in between and no additional inoculation for the second period. Nodule occupancy of the introduced S. meliloti in non-sterile soils ranged from 30 to 60% for the first period and 85 to 100% for the second period. Our results suggest that survival and persistence of S. meliloti was enhanced by alfalfa cultivation and increased soil fertility, but impaired by mutation of acid phosphatase genes regardless of phosphorus nutritional levels. Moreover, inoculation with genetically modified S. meliloti strain 104A14 promoted indigenous bacterial growth in soil (increased bacterial population from 1.4×10⁶ to 4.3×10⁶ cells g⁻¹ soil), but not the growth of fungi and yeast. However, inoculation of the wild-type S. meliloti or genetically modified mutants did not result in significant changes in microbial community structure as indicated by EP indices and ratios of r/K strategists.     

嗜水气单胞菌转录调控蛋白基因(ahyR)突变株的构建及特性分析

根据GenBank公布的嗜水气单胞菌(Aeromonashydrophila,Ah)转录调控蛋白基因ahyR序列设计1对特异性引物,利用PCR方法扩增AhJ-1株ahyR的部分片段。PCR产物克隆入pMD18-T载体,经序列测定后定向连接入含有卡那霉素抗性(Kanr)基因的自杀性质粒pJP5603中,构建重组质粒pJP-ahyR。将鉴定为阳性的重组质粒转化嗜水气单胞菌J-1株感受态细胞,获得1突变株细菌。PCR方法鉴定该突变株为J-1株ahyR基因同源突变株,命名为J-1△ahyR。与J-1株相比,突变株的外膜蛋白图谱和部分生化特性发生改变;胞外蛋白酶、淀粉酶、DNA酶、溶血素等几种主要毒力因子同时丧失;致病力明显降低,对小白鼠的半数致死量大于1.0×108CFU(colonyformingunits)。该研究为ahyR基因功能的探讨及嗜水气单胞菌弱毒疫苗的研制奠定了基础。     

Variation in symbiotic characters of alfalfa cultivars inoculated with Sinorhizobium meliloti strains

In this paper the interactive effects of different Sinorhizobium meliloti strains and alfalfa cultivars on the host plant growth and nodulation were evaluated. In the experiments, six alfalfa cultivars (Giza-1, Ismailia-94, Kometa, Radius, Luzella and Legend) as well as five S. meliloti strains (ARC-1, ARC-2, A₂, TAL and L5-30) were included. The alfalfa cultivars, Giza-1 and Ismailia-94, exhibited the highest average dry forage weight with all studied strains. Generally, plants inoculated with the S. meliliti ARC-2 strain produced significantly higher shoot and root dry weights over all evaluated cultivars. The strain L5-30 of S. meliloti produced the highest nodule number with all the studied alfalfa cultivars whereas, among tested cultivars, N₂-fixing Luzella showed the highest nodulation with the majority of rhizobial strains. Analysis of correlation coefficients for the studied alfalfa-rhizobium associations revealed positive and significant relationships of shoot dry weight with root dry weight. In contrast, nodule number per plant correlated negatively with forage dry weight of plants. It was also found that nodule number per plant is not a promising trait with which to select the most effective N₂-fixing rhizobium-legume systems; selection of such symbiotic associations should be done on the basis of shoot and root dry weights.     

水稻淡黄叶突变体pyl3的鉴定和基因定位

利用⁶⁰Co-γ辐射诱变籼稻骨干恢复系川恢907(R907),从突变体库中筛选获得一份淡黄叶突变体pyl3 (pale yellow leaf mutant 3)。为揭示pyl3突变体淡黄叶表型的调控机制,本研究对该突变体和野生型进行表型鉴定、主要农艺性状和基因定位分析。结果表明,从苗期开始pyl3突变体整个生育期表现淡黄叶表型;与野生型相比,pyl3在苗期叶片中的叶绿素和类胡萝卜素含量显著降低;成熟期pyl3的株高、穗长、穗粒数和结实率等农艺性状指标均显著下降。遗传分析结果表明,pyl3突变体的表型由一对隐性核基因控制。利用分子标记连锁分析的方法将该基因初步定位于第3号染色体上InDel标记M4和M6之间,物理距离约为3.2 Mb。进一步基于BSA全基因组重测序和Sanger测序分析发现,pyl3突变体在定位区间内编码Mg-原卟啉Ⅸ螯合酶CHLI亚基的基因编码区第1 034位碱基C突变为碱基T,导致编码的第345位的半胱氨酸(Cys)突变为酪氨酸(Tyr)。pyl3突变体中携带的CHLI^pyl3是已报道黄绿叶基因chl9/chli的新等位基因。qRT-PCR分析结果显示,pyl3突变体中叶绿素合成和光合作用相关基因的表达发生变化。本研究为进一步解析CHLI基因调控水稻叶色的分子机制提供了新的遗传材料和理论依据。     

Response of alfalfa (Medicago sativa L.) to single and mixed inoculation with phosphate-solubilizing bacteria and Sinorhizobium meliloti

The objectives of this work were to phenotypically and genetically characterize alfalfa rhizosphere bacteria and to evaluate the effect of single or mixed inoculation upon nodulation and biological nitrogen fixation. Thirty-two strains showed tricalcium phosphate solubilization ability, and two of them caused bigger or equal solubilization halos than the control strain P. putida SP22. The comparison of the 16S ribosomal DNA sequences indicated that these strains are phylogenetically related to Bacillus spp. and Pseudomonas spp. A beneficial effect of both isolates on alfalfa growth was observed in coinoculation assays. Pseudomonas sp. FM7d caused a significant increase in root and shoot dry weight, length, and surface area of roots, number, and symbiotic properties of alfalfa plants. The plants coinoculated with Sinorhizobium meliloti B399 and the Bacillus sp. M7c showed significant increases in the measured parameters. Our results indicating that strains Pseudomonas sp. FM7d and Bacillus sp. M7c can be considered for the formulation of new inoculants.     

水稻淡黄叶突变体xws的光合特性与基因定位

为了解析水稻叶绿体发育和高光效育种中光合作用的机理,本研究以不育系P88S群体中发现的一株淡黄叶自然突变体xws为试验材料,对该突变体进行表型鉴定和光合特征分析,并对其进行精细定位和候选基因分析。结果表明,xws在整个生育期叶片、茎和穗均呈淡黄色。与野生型相比,xws叶片、茎和穗的叶绿素a、叶绿素b和类胡萝卜素含量均显著下降。透射电镜观察结果发现,与野生型相比,xws嗜锇小体消失,类囊体数量减少。xws的净光合效率、气孔导度、蒸腾速率、胞间CO₂浓度和叶绿素荧光参数与野生型相比均降低。以xws与R032杂交的F₂群体作为定位群体,将基因精细定位至水稻第3号染色体分子标记WY242和WY119之间,其物理距离为51.7 kb。本研究结果为进一步研究水稻叶绿体发育,从而提高水稻光合作用和分子调控网络途径提供了理论支撑。     

Survey of genes identified in Sinorhizobium meliloti spp., necessaryfor the development of an efficient symbiosis

The symbiosis between the soil bacteria Rhizobium, Sinorhizobium, Azorhizobium, Mesorhizobium or Bradyrhizobium and leguminous plants is characterised by a specific multistep signal exchange. Only when a compatible rhizobial strain encounters its leguminous host, nodules will be formed on the roots of the host. During infection of this nodule, the microsymbiont evolves into a bacteroid form which, when provided with plant-derived carbon sources, is able to convert atmospheric nitrogen to ammonia that subsequently is supplied to the plant. The developmental programme underlying nodule organogenesis and functioning has been studied intensively for several decades. In this review, several observed plant phenotypes resulting from an ineffective symbiosis between plants and mutant rhizobial strains are represented. Besides the influence of the bacterial nodulation, nitrogen fixation and surface polysaccharide genes on symbiosis, the role of other genes important for the formation of effective nitrogen fixing nodules will be explained.     

山羊痘病毒P32基因跨膜区缺失载体的构建与表达*

P32基因是山羊痘病毒的主要免疫原性基因,含有跨膜区,用一般的诱导方法进行诱导表达,表达量较低。本文尝试用重组PCR消除跨膜区,连接入pGEX-6P-1载体,构建pGEX-6p-s重组表达载体,再用IPTG进行诱导表达,SDS-PAGE结果发现,在58KDa处检测到蛋白,与预期目的蛋白位置相符,用山羊痘标准阳性对表达产物进行Western检测,表达产物可被山羊痘标准阳性血清识别。表达产物以融合蛋白的形式存在,融合蛋白出现在沉淀中,以包涵体的形式存在,用Bandscan软件对所表达的蛋白进行分析发现,表达产物约占菌体蛋白的29.4%。     

肝靶向穿膜肽与家蝇天蚕素基因的融合及其分子特征分析

本研究利用改进SOE-PCR技术构建肝靶向穿膜肽（HTPP）与家蝇天蚕素（MDC）融合基因并对其分子特征进行了预测和分析.结果表明：成功融合了HTPP与MDC,并构建了HTPP-MDC融合基因的克隆重组质粒HTPP-MDC/pMD20-T.PCR和KpnⅠ/Hind Ⅲ双酶切结果显示获得与预期大小一致的基因片段,测序结果显示获得的基因序列没有发生突变,与预期完全一致.分子特征分析表明,该融合基因编码60个氨基酸,分子量为6 516.2 Da,理论等电点为 9.31,二级结构主要由α-螺旋、无规则卷曲、延伸链和β-转角组成.研究结果为HTPP-MDC后续的功能研究奠定了基础,同时也为应用SOE-PCR技术构建融合基因提供了有益借鉴.