亚洲百合遗传转化受体系统的建立

以亚洲百合"精粹"(Lilium Asiatic 'Elite')的鳞片、再生形成的叶片和鳞片叶为外植体,进行了不定芽的诱导分化、试管苗小鳞茎诱导、卡那霉素抗性筛选等研究,建立了亚洲百合遗传转化受体系统.结果表明:鳞片诱导最佳分化培养基为MS 6-BA 2.0 mg/L NAA 0.2 mg/L,试管苗小鳞茎诱导培养基为MS IBA 0.5 mg/L 70 g/L蔗糖,试管苗叶片分化培养基为MS TDZ 1.0 mg/L NAA 0.2 mg/L,试管苗鳞片叶分化培养基为MS 6-BA 2.0 mg/L 2,4-D 0.1 mg/L.抗生素敏感性试验表明,百合叶片的卡那霉素选择压为80 mg/L,鳞片叶为115 mg/L.     

蛋白酶抑制剂基因真空渗入法转化不结球白菜获得抗虫性材料

菜青虫及小菜蛾是危害白菜生产的主要害虫，为了获得新的抗虫白菜种质，以生长45天、花苔抽出时的“49菜心”Brassica rapa ssp．chinensis var．utilis Tsen et Lee为材料，通过根癌农杆菌介导的真空渗入法，将马铃薯蛋白酶抑制剂基因（pin Ⅱ）导入该植物基因组中。在142株处理株的后代种子中，采用除草剂Basta筛选，获得41株生长良好的抗性植株。经氯酚红试验、PCR以及植物基因组Southern杂交鉴定，证实目的基因已经整合入植物基因组中。对小菜蛾Plutella xylostella L．进行了转基因材料离体叶片的连续饲喂，调查了自1龄幼虫至化蛹、羽化的动态变化。结果表明。受试的转基因植株对小菜蛾的生长发育有明显的抑制作用。     

欧美杨细菌性溃疡病菌双组份系统反应调节基因LqRR2的功能研究

由Lonsdalea quercina subsp.populi引起的欧美杨溃疡病于2006年在国内首次发现,不同于其它病原菌造成的杨树溃疡病,该病害对欧美杨速生林的生长造成毁灭性破坏,已造成了严重的经济损失,该病原菌的致病分子机制尚不清楚。双组分系统是细菌重要的信号传递通路,在细菌的生长繁殖、逆境胁迫应答、环境适应以及病原菌致病过程中发挥重要作用。开展双组份系统研究将有助于解析欧美杨细菌性溃疡病菌的致病机制。本研究鉴定了欧美杨细菌性溃疡病菌L.quercina双组份孤儿反应调节基因LqRR2,并对其生物学功能进行了研究。通过同源重组获得了LqRR2基因的缺失突变体△LqRR2。表型测定结果显示,与野生型菌株相比,突变体△LqRR2在半固体培养基上的游动能力显著减弱,对欧美杨‘107杨’枝干的毒性也显著降低。但是,突变体的生长速率、生物膜形成能力以及胞外多糖产量较野生型无显著差别。此外,荧光定量PCR分析结果显示,游动性相关基因flg B、flg C和flg E的表达量在突变体中明显降低。综上所述,双组份调节蛋白编码基因LqRR2是欧美杨细菌性溃疡病菌L.quercina维持病原菌游动性和全毒性所必需的。     

稻瘟病菌转化体系的建立及3-磷酸甘油醛脱氢酶基因(gpd)的克隆

 通过构建cosmid质粒,建立了稻瘟病菌(Magnaporthe grisea)的遗传转化体系和基因组文库;并通过cDNA文库和PCR技术相结合的方法,克隆了稻瘟病菌3-磷酸甘油醛脱氢酶基因(gpd),进行了基因的核苷酸序列分析。     

编码桔小实蝇抗阿维菌素的GABA受体基因5'端序列片段克隆与分析

室内培育出桔小实蝇Bactrocera dorsalis(Hendel)的敏感品系和抗阿维菌素品系(AvR90),抗性品系对阿维菌素的抗性是敏感品系的113.12倍。克隆出桔小实蝇抗阿维菌素品系和敏感品系中编码GABA受体基因5'端序列1053 bp,发现AvR90品系与敏感品系的该片段的碱基有一个差异(A496T),但其推导的氨基酸相比没有位点发生突变。     

应用DNA标记定位水稻的抗稻瘟病基因

 从水稻分子遗传图谱选取177个RFLP标记,比较以红脚占为抗源,感病品种IR24为轮回亲本所构建的近等基因对K80R和K79S之间的多态性表现。发现了一些可能与稻瘟病抗性基因连锁的阳性标记。在(K80R×K79S)的F₂群体中,经稻瘟病菌ZB₁小种接种,110株为抗病,33株为感病,用总共10个阳性标记与F₂群体中每个单株的DNA杂交,发现抗病基因与第12染色体上的标记RG81、RG869和RZ397共分离;检测F₃株系的抗病性分离情况,确定F₂植株的抗病性基因型,计算出抗病基因与分子标记的遗传距离,将该基因定位在第12连锁群上。应用近等基因池DNA和随机引物,经PCR扩增和共分离分析,建立了二个RAPD片段与抗病基因紧密连锁。     

氯虫苯甲酰胺对小菜蛾鱼尼丁受体基因mRNA表达量的影响

采用实时荧光定量PCR研究比较了氯虫苯甲酰胺、甲氨基阿维菌素苯甲酸盐和溴虫腈对小菜蛾鱼尼丁受体基因mRNA表达量的影响,研究分析了低剂量氯虫苯甲酰胺连续处理和间断处理对小菜蛾鱼尼丁受体基因表达量的影响,以及对氯虫苯甲酰胺产生抗性的小菜蛾与敏感小菜蛾鱼尼丁受体基因表达量的差异。结果表明:氯虫苯甲酰胺可提高小菜蛾鱼尼丁受体基因表达量,而甲氨基阿维菌素苯甲酸盐和溴虫腈对其表达量均无影响;0.05和0.01 mg/L的氯虫苯甲酰胺均可使小菜蛾鱼尼丁受体基因表达量增加;对氯虫苯甲酰胺产生抗性的小菜蛾鱼尼丁受体基因表达量是敏感小菜蛾的5.79倍。研究结果有助于阐明氯虫苯甲酰胺的作用机理,并可能为筛选作用于鱼尼丁受体的新化合物提供参考。     

抗菌蛋白AP1编码基因对马铃薯的转化及其介导的青枯病抗性研究

根据已知马铃薯抗青枯菌蛋白AP1编码基因序列,合成了特异性引物,经PCR扩增、酶切以及连接、转化等分子操作,成功地获得了AP1编码基因植物表达载体pHap1,该载体具有-2E-P35S-12-ap1-结构。以电激法将pHap1导入根癌农杆菌LBA4404,用叶盘法转化马铃薯青枯病感病品种Mira、中-5-1及金冠试管苗,分别获得了卡那霉素(kan)抗性再生苗。经PCR、Southern、Northern检测,表明ap1基因已成功整合到转基因植株染色体上并正确表达。对Mira品种的转基因植株进行了青枯病抗病性鉴定,结果表明,转基因植株的抗病性比非转基因植株对照明显提高,发病或出现萎蔫症状的时间延迟、病情指数降低。     

基于原生质体转化的甘蔗梢腐病菌YN41基因敲除体系的构建

基因敲除技术日益成为基因功能研究的重要手段,为了深入研究甘蔗梢腐病菌致病基因的功能,构建了梢腐病菌YN41菌株的基因敲除体系。本研究构建了基于线性DNA的同源重组基因敲除技术,融合PCR构建敲除盒即带有潮霉素基因标记的线性DNA融合片段,配置0.05 g·mL^-1溶壁酶+0.01 g·mL^-1崩溃酶的复合酶液28℃酶解3 h获得了高产量的原生质体,利用聚乙二醇(PEG)介导的原生质体的转化,PCR鉴定技术和酶切鉴定技术对转化子进行鉴定,荧光定量PCR分析和Southern Blot方法进一步对基因缺失突变体进行验证,生物学表型(菌落形态、生长速率、产孢量、生物量和致病力)验证了PK基因敲除体系的成功构建。28℃酶解3 h获得了2×10⁷个·mL^-1原生质体;对编码丙酮酸激酶的PK基因进行了基因敲除验证,获得了纯合敲除突变体;荧光定量PCR检测转录水平无表达;Southern Blot结果显示使用PK基因探针杂交,基因缺失突变体无条带,野生型杂交到目的条带;生物学表型验证了PK基因敲除突变体对比野生型...     

烟碱乙酰胆碱受体及其与新烟碱类相互作用的研究进展

对烟碱乙酰胆碱受体(nAChRs)的结构与功能、配体结合部位、门控机理以及与新烟碱类的相互作用进行了综述,并对nAChRs亚基基因突变和敲除对新烟碱类和多杀菌素敏感性的影响进行了讨论。nAChRs在脊椎动物和昆虫的胆碱能突触的快速神经传递中起着重要作用,其在昆虫中仅存在于中枢神经系统(CNS)中,而在脊椎动物中同时存在于CNS和神经肌肉连接处。nAChRs是新烟碱类杀虫剂、多杀菌素和杀螟丹的作用靶标。肌肉和CNS中的nAChRs是一个由两个α和三个非α(β,γ和δ)亚基组成的异数五聚体,该受体主要有三部分:一个在细胞外发现的区域(胞外区)、一个位于膜内的区域(跨膜区),另一个是位于细胞内的区域(胞质区)。每个亚基(从N-C端)都具有一个包含乙酰胆碱(ACh)结合部位的细胞外结构域;4个跨膜结构域(M1~4),其中M2的大部分氨基酸位于离子通道的内壁;一个胞质噜扑(loop)和一个胞外C端。通道门位于孔道内的疏水区。ACh结合部位位于天然和功能受体的两个亚基的界面,是由一个亚基的3个噜扑(A-C)和另一个亚基的3个噜扑(D-F)构成。每当受体与ACh(或其他激动剂)分子结合时,M2 α螺旋体的构象发生改变,使通道开启,处于阳离子传导状态,直至一个或两个激动剂分子从结合口袋解离,通道才关闭。如果激动剂一直存在,并反复结合,则通道处于脱敏状态。nAChRs与新烟碱类的各种选择性作用取决于新烟碱类的结构以及nAChRs的亚基组成。     

D-Alanine-D-Alanine Ligase Gene (ddl) of Erwinia chrysanthemi Strain EC16 I. Isolation and Gene Dosage Effect on Pectate Lyase Synthesis

New regulatory gene for pectate lyase (Pel) production of Erwinia chrysanthemi strain EC16 was searched by observing the gene dosage effect of each cosmid library in Pel production. From this survey, a cosmid clone, p5A, had reduced in Pel production under both inducing and non-inducing conditions and caused less tissue maceration of potato tubers. The 2.64-kb HindIII fragment from p5A was found to be responsible for this phenotype and to contain one major ORF consisting of 1107 nucleotides, which had homology with ddlA (49.6%) and ddlB (49.6%) of Escherichia coli and with ddlA (44.6%) of Salmonella typhimurium. These genes had been shown to encode D-alanine-D-alanine ligase (Ddl), an enzyme which is involved in the synthesis of peptidoglycan. This ORF encodes 368 amino acid residues and its molecular weight is estimated to be 43 kDa. This ORF of EC16 could complement ddl deficient mutant of Escherichia coli. Received 15 May 2002/ Accepted in revised form 20 June 2002     

sfp基因转化增强了Bacillus subtilis 168的定殖能力和对黄瓜茎内枯萎病菌的抑制作用

枯草芽胞杆菌B006是一株防治黄瓜枯萎病的高效菌株,可产生脂肽类抗生素表面活性素surfactin和芬枯草菌素fengycin。为深入了解surfactin在根际的作用,本研究通过同源重组的方法将生防芽胞杆菌B006菌株中编码4'-磷酸泛酰巯基乙胺转移酶Sfp的sfp基因整合到不产生surfactin的模式菌株B.subtilis168(B168)基因组中,获得了重组菌株B168S。重组菌株B168S在1%淀粉平板上不水解淀粉,在5%血平板上产生透明溶血圈;HPLC-ESI-MS检测表明菌株B168S在牛肉膏蛋白胨培养液中培养48 h后可产生surfactin。平板对峙试验表明菌株B168S在黄瓜根分泌物培养基上对Fusarium oxysporum f. sp.cucumerinum(Foc)菌丝生长有一定的抑制作用,抑制率R2/R1为1.22;平板菌落计数法测定表明,将菌悬液(10⁶ cfu/mL)浸泡90 min的黄瓜种子播种于无菌石英砂3 d后,菌株B168S在黄瓜根基部和根尖的定殖量分别为菌株B168的2~3倍和9~10倍;温室盆栽试验表明,黄瓜苗用浓度为106 cfu/mL的B168S和B168菌悬液蘸根后,移栽到接种Foc孢子(10⁵孢子/g)的病土中,移栽后第3周菌株B168S和B168处理的防效分别为21.1%和17.9%;对不同高度黄瓜茎中Foc的组织分离结果表明,菌株B168S处理后Foc在黄瓜茎内蔓延的相对高度低于菌株B168和Foc处理。上述结果进一步明确了surfactin可明显促进芽胞杆菌在黄瓜根部的定殖,并通过抑制枯萎病菌的侵染和在黄瓜茎中的蔓延增强对黄瓜枯萎病的防治效果。     

Transformation of carrots with mutant acetolactate synthase for Orobanche (broomrape) control

Parasitic Orobanche spp are major constraints to vegetable crop production in the Mediterranean basin (to eastern Europe) and in localized places in India, China and the USA. Transgenic target-site herbicide resistance (eg, to acetolactate synthase inhibitors) allows for movement of unmetabolized herbicide through the crop to the photosynthate sink in the parasite, as well as through the soil. We report the successful engineering of a mutant acetolactate synthase (ALS) gene into carrot, allowing control of broomrape already in heterozygotes of the first back-crossed generation, by imazapyr, an imidazolinone ALS inhibitor. It is expected that homozygotes will have higher levels of resistance.     

侵染百合的黄瓜花叶病毒亚组Ⅱ分离物cp基因克隆和序列分析

 从云南大理的东方型百合上得到黄瓜花叶病毒分离物(CMV-DL), ELISA检测初步确定为CMV亚组Ⅱ分离物, 设计并合成CMV亚组Ⅱ的特异引物, RT-PCR扩增得到1条约800 nt的特异片段, 经克隆及序列测定, 该片段长828 nt, 包含的外壳蛋白(CP)基因由657 nt组成。将该分离物的cp基因与其它14个CMV分离物进行同源性比较, 在核苷酸水平上与CMV亚组I和亚组Ⅱ的同源性分别为76.8%~78.1%和98.6%~99.2%;在氨基酸水平上与CMV亚组I和亚组Ⅱ的同源性分别为82.0%~84.3%和95.9%~100.0%。结果表明CMV-DL为CMV亚组Ⅱ成员。     

Isolation and Characterization of a New Virulence Gene (abvA) of Agrobacterium tumefaciens

A mutant (M-1) was isolated by transposon (Tn5) insertion mutagenesis of Agrobacterium tumefaciens (strain A-208, C58 chromosome, nopaline type T37 pTi, virulent). The M-1 mutant exhibited a complete avirulent phenotype on Kalanchoe daigremontiana leaf and Kalanchoe pinnata stem but a very attenuated virulent phenotype on root of Daucus carota. The mutant had one insertion of Tn5 in pTi. A wild-type target segment (2.3 kb) that included the site of Tn5 insertion in M-1 mutant was cloned. Introducing the 2.3 kb segment into M-1 complemented completely the avirulent phenotype, producing galls as big as strain A-208. The 2.3 kb segment was sequenced, identifying three open reading frames, ORF 1 (354 bp), ORF 2 (261 bp) and ORF 3 (801 bp) in the segment. A Tn5 was inserted between the third and fourth nucleotide of ORF 1 in M-1. The ORF 1 had no homology to any reported genes and thus was named the abvA gene. The ORF 3 had the high homology (identities 44%, positive 68%) to the gene of the sarcosine oxidase β subunit (accession no. sp/P40875). Introduction of the DNA segment (743 bp) containing the abvA gene and its promoter region into M-1 partially complemented the avirulent phenotype of the mutant, producing galls smaller than strain A-208. The abvA gene was distributed not only on nopaline-type pTi (T37) but also on octopine-type pTi (A6NC) and chromosome (C58) of A. tumefaciens. M-1, being avirulent on K. daigremontiana and K. pinnata, had a Tn5 insertion only in the abvA gene on pTi but not in the abvA gene on the chromosome, implying that the abvA gene on the chromosome in strain A-208 is not functional. A binary vector, pIG121-Hm, containing the β -glucuronidase (GUS) gene with an intron was introduced into M-1, which was then applied to leaves of K. daigremontiana to assay GUS activity for monitoring T-DNA transfer to the host nucleus. High GUS activity comparable to that in strain A-208 was detected in M-1 in spite of its inability to induce galls, suggesting that M-1 can transfer T-DNA into the host nucleus, but cannot integrate it into the chromosome. Received 25 October 2000/ Accepted in revised form 28 December 2000     

Involvement of gacA Gene in the Suppression of Tomato Bacterial Wilt by Pseudomonas fluorescens FPT9601

Pseudomonas fluorescens FPT9601, a plant growth-promoting rhizobacterium (PGPR) isolated from tomato rhizosphere, can protect tomato (Lycopersicon esculentum Mill) from bacterial wilt disease caused by Ralstonia solanacearum. This strain produces antibiotics 2,4-diacetylphloroglucinol (2,4-DAPG) and hydrogen cyanide (HCN). It also produces proteases and uncharacterized siderophores (Sid). A mutant strain SM2214, obtained by Tn5 insertion, did not produce 2,4-DAPG, HCN or proteases, but overproduced Sid. Marker-exchange mutagenesis confirmed that a single transposon insertion caused the multiple phenotypic changes of this mutant. Complementation of the mutant with a 1.3-kb DNA fragment that was amplified from genomic DNA of the wild-type P. fluorescens strain by PCR could restore the lost functions of the mutant strain. Nucleotide sequencing revealed that the fragment contained a 642-bp open reading frame (ORF) highly homologous to the regulator responser gene gacA. The in vitro anti-bacterium test and plant protection experiment under greenhouse conditions indicated that the gacA gene played an important role in the suppression of tomato bacterial wilt disease. Received 20 November 2000/ Accepted in revised form 19 January 2001     

36个小麦审定和区试品种(系)的抗白粉病基因推导

明确我国当前小麦审定和区试品种含有的抗白粉病基因, 可为这些品种在小麦抗病育种中的应用及品种合理布局和轮换提供依据。本研究采用24个不同毒性的小麦白粉菌菌株对36个小麦审定和区试品种(系)进行抗白粉病基因推导, 参试品种(系)与46个已知抗病基因小麦品种(系)抗谱比较的结果表明, 11个小麦审定品种中有5个品种对所有供试白粉菌菌株表现抗性, 结合亲本溯源, 推测其中有3个品种可能携有Pm21基因; 另外6个审定品种中有5个品种可能含有抗病基因Pm2, 且其对应的亲本或亲本组合中含有抗病基因Pm2。25个区试品种(系)中有3个可能含有Pm21, 10个含有Pm2, 1个含有Pm2+6,2个含有Pm4b,1个含有Pm8。另外, 参试的36个品种(系)中还有9个品种(系)和已知基因品种抗谱存在一定差异。总体上, 推导出已知基因的品种以含有Pm2基因的品种最多, Pm21基因的品种次之, 建议在生产上加强对Pm21基因品种(系)特别是已审定的携有Pm21基因品种的推广和应用, 应该注意一些省份在育种和生产上应慎用或少用含Pm2基因的品种(系)。     

2个适于水稻条斑病菌致病相关基因转录表达分析的启动子探针载体的构建

 水稻条斑病菌(Xanthomonas oryzae pv. oryzicola, Xoc)为稻黄单胞菌种下的致病变种,引起细菌性条斑病(bacterial leaf streak, BLS),对水稻安全生产构成严重威胁。为准确在水稻条斑病菌中进行致病相关基因的转录表达和调控分析,本研究构建了包含终止子、gusA报道基因和多克隆位点等启动子探针元件的载体pUTG01和pUTG14。选取Xoc的hrpF启动子,构建在pUTG01载体上,将其导入hrpX突变体RΔhrpX中,GUS活性测定结果显示,hrpF基因的表达显著减低,验证了hrpF受HrpX正调控,证实该载体可有效进行基因的转录表达分析;通过双质粒兼容共存策略,在hrpX突变体中同时实现了hrpX基因的功能互补和通过GUS活性定量测定hrpF基因的转录表达分析。该载体系统的建立,为后续分析稻黄单胞菌致病相关基因的表达调控提供了有效的工作系统。     

结球甘蓝抗根肿病SSR分子连锁图谱的构建

结球甘蓝(Brassica oleracea L.var.capitata)简称甘蓝,是我国重要的蔬菜作物之一.甘蓝根肿病是由芸苔根肿菌(Plamodiophora brassicae Woron.)侵染引起的一种世界性真菌病害.近年来,该病在我国的发病面积急剧增加,造成甘蓝产量和品质大幅度降低,选育抗病品种成为防治根肿病的重要途径之一.