含双病原物诱导启动子无选择标记转基因烟草的获得

Transgenic plants are controversial for their edible and environmental security. Marker-free transgenic plants can be produced by the construction and transformation of plant expression vectors carrying twin T-DNAs. The construction of plant expression vectors harboring twin T-DNAs and two pathogen-inducible promoters was previously reported. These vector plasmids were introduced into tobacco plants and the transgenic tobacco plants were obtained. In this paper we report that T₁ transgenic tobacco seedlings were produced through classical genetics approach. Analysis of the seedlings demonstrated that some of them were marker-free lines. The segregation of exogenous genes in T₁ transgenic tobacco seedlings was tested by using two methods. Firstly, the ability of resistance to kanamycin was analyzed in T₁ transgenic tobacco seedlings from 14 transgenic plant lines. It was found that the segregation ratio of NPTII genes met well with Mendel’s law in 13 transgenic tobacco lines. So it was deduced that NPTII gene was as a single copy integrated into one of the homologous chromosomes. Then the NPTII genes and uidA genes of 130 T₁ transgenic seedlings were detected from the above 13 tobacco lines. The results showed that uidA genes were only detected in 20.77% of the seedlings, NPTII genes were solely detected in 22.31% of the seedlings, but both exogenous genes were in 53.85% of the seedlings. The segregation ratio of the two genes was consistent with the law of independent assortment (9∶3∶3∶1). These results suggested that the selective marker gene had no linkage with the reporter gene and they were segregated independently in the T₁ transgenic tobacco plants. This method, as compared with traditional backcross, is confirmed a more easy and rapid way to eliminate the antibiotic resistance gene used as a selective marker in transgenic plants.     

Parasiticein诱导烟草细胞凋亡的形态及生化特征

【目的】研究烟草细胞对parasiticein的反应,了解parasiticein诱导烟草细胞的PCD的发生机制。【方法】用浓度约100 nmol•L-1的parasiticein处理烟草悬浮细胞,相差显微镜下观察细胞的形态变化。【结果】发现烟草细胞的细胞膜收缩,与细胞壁间形成空隙,原生质浓缩等;用trypan blue 染色后观察到类似现象。对诱导处理的悬浮细胞经DAPI（4,6-diamino-2-phenylindole）染色后,通过荧光显微镜观察,发现细胞核先后表现染色质凝集、形成颗粒状等特征;对处理的悬浮细胞提取基因组DNA并进行琼脂糖凝胶电泳,可观察到梯状的DNA条带（DNA laddering）。研究还发现,parasiticein诱导的烟草细胞死亡可被蛋白激酶信号通路抑制剂K252-a部分抑制。【结论】parasiticein诱导的烟草细胞死亡具有细胞凋亡的典型形态及生化特征,是一种主动的细胞死亡过程。     

烟草抗赤星病诱导剂SRS2的田间应用

作者在温室、田间上区和大田进行应用示范试验,明确了诱导剂ＳＢＳ２对烟草赤星病的控制作用。室内测定结果表明,ＳＲＳ２与菌核净防治效果相当,均接近１００％。１９９４年在山东沂南作了小区试验,１９９４、１９９５年在山东和安徽８县（市）进行了应用示范试验,结果表明,诱导剂ＳＲＳ２能够较好地控制烟草赤星病,最高可达８５％,平均防治效果为６２．７％,与菌核净相比,ＳＲＳ２的防治效果平均高出２．７％。     

生姜茎基腐病病原拮抗细菌的筛选与鉴定

从生姜根际土壤分离得到62个细菌分离物。以姜茎基腐病病原菌(Pythium myriotylum)为指示菌,共选出7株对姜茎基腐病菌有良好拮抗效果的细菌菌株。经过形态学观察、生理生化测定,16S rDNA序列及系统发育分析,结合《常见细菌系统鉴定手册》等,X-11、JK-14、SF-19、SF-23等4株初步鉴定为芽孢杆菌属(Bacillus spp.),JK-19、JK-28、JK-25 3株鉴定为类芽孢杆菌属(Peanibacillus spp.)。SF-19、SF-23、JK-25、JK-19、JK-28、X-11和JK-14的16S rDNA序列的GenBank登录号分别为JQ283970、JQ283971、JQ283972、JQ283973、JQ283974、JQ283975、JQ283976和JQ283977。进一步分析表明,7株拮抗菌对Rhizoctonia solani、Fusarium graminearum、Verticillium dahliae等多种类型的病原真菌有较好的抑制作用;显微观察结果表明,拮抗菌可导致病菌菌丝细胞顶端膨大、菌丝体畸形、分隔增多等变化。     

利用GICA-PCR快速检测瓜类果斑病菌

瓜类果斑病菌(Acidovorax avenae subsp.citrulli,Aac)是瓜类作物上重要的病原细菌,为我国进境植物检疫性有害生物。胶体金免疫层析试纸条方便快捷,应用广泛。该方法使用不当会出现假阳性问题,仅适用于病原菌的初筛检测。本研究将Aac胶体金免疫层析方法(GICA)与PCR技术相结合,建立了GICA-PCR检测方法。检测结果表明,该方法在蛋白与核酸2个层面上从发病西瓜叶片上检测到瓜类果斑病菌,有效解决了试纸条检测的假阳性问题,提高了瓜类果斑病菌检测的准确性,值得推广应用。     

烟草赤星病菌糖蛋白激发子诱导烟草抗病防卫反应

用来自赤星病菌弱毒株的糖蛋白激发子处理烟草，能明显抑制烟煤草体内过氧化氢酶（CAT）的活性，增加组织中H2O2的含量，诱导后1dCAT活性就已开始下降，至第6天下降到最低，以后又逐渐恢复，而H2O2含量的变化趋势与CAT活性的变化相反，随CAT活性的降低，组织中H2O2含量逐渐增加，在诱导后第10天达到最大值，激发子处理烟草还可以诱导碱性PR蛋白基因PR1b的表达；同时，烟草对赤星病强毒株的抗性逐渐增强，以诱导后第10天接种抗性最强，且这种抗性是系统性的，水杨酸（SA）处理后CAT及H2O2的变化与上述趋势类似。     

串联双病原物诱导启动子驱动基因表达的特性

 在植物抗病基因工程中,目标基因可控的高效表达是一重要目标。在前期的研究中,我们将串联的病原物高度特异性诱导启动子EAS4(EP)和hsr203J(HP)转入烟草,uidA基因在相应病原物或激发子诱导后可被驱动表达。为进一步研究此串联双启动子驱动的基因表达特性,采用荧光法对GUS诱导表达活性进行了定量检测。结果发现,双启动子表现较高的诱导表达活性,双启动子与单启动子的活性差异均达极显著水平。研究还发现,两个启动子串联的顺序对其活性强度和时间动态有影响,双启动子HP+EP的诱导表达高峰出现在诱导后6~10 h,而EP+HP的活性高峰出现在诱导后8~14 h。同时,初步分析了串联的EP和HP协同促进基因高水平表达的原因。串联的双病原物特异性诱导启动子不但可响应较广谱的病原物的诱导表达,而且可协同促进基因的高效表达,因而在植物抗病基因工程中具有广阔的应用前景。     

烟草根际溶磷解钾细菌的筛选鉴定及对烟草的促生作用

本试验以山东潍坊烟田土壤为研究对象,对其根际溶磷解钾细菌进行筛选鉴定并分析该类细菌对烟草生长的影响。结果显示:(1)从山东潍坊烟田土壤样品分离得到34个细菌分离物。以烟草黑胫病菌(Phytophthora parasitica)和烟草青枯病菌(Ralstonia solanacearum)为指示菌,筛选出对2种病原菌有良好拮抗效果的菌株13株。(2)测定发现8株菌株具有良好的溶磷能力,菌株培养液中可溶性磷含量最高为241mg/L。5株菌株具有解钾能力,菌株培养液中速效钾含量最高为13.95 mg/L。5株菌株具有产IAA能力,最高IAA产量为104.71μg/mL。(3)菌株H1-2、3183可明显促进烟草生长,其浇灌后的烟草地上部和地下部鲜重均高于对照。(4)经16S r DNA序列系统进化分析得出,菌株3183和1441为贝莱思茅芽孢杆菌(Bacillus velezensis),菌株H1-2为多黏类芽孢杆菌(Peanibacillus polymyxa),菌株G2-7和D2为伯克霍尔德氏菌(Burkholderia spp.)。     

核黄素诱导烟草悬浮细胞酚类物质和木质素的积累

 【目的】阐明核黄素对烟草悬浮细胞苯丙氨酸解氨酶(phenylalanine ammonia-lyase,PAL)、过氧化物酶(peroxidase,POD)活性以及抗病相关的酚类物质、木质素等次生代谢产物含量的影响。【方法】1 mmol?L-1的核黄素处理烟草悬浮细胞,用生物化学、细胞化学和高效液相色谱(high performance liquid chromatography,HPLC)等方法测定苯丙烷代谢有关的生理和生化指标。【结果】核黄素处理后,PAL和POD活性明显升高,处理12 h 和24 h后酶活性分别达到峰值;荧光观察细胞壁积累了更多的酚类物质,胞内和胞外scopoletin(7-羟基-6-甲氧基香豆素)含量明显升高,在处理12 h后都达到峰值,分别是对照的3.6和3.9倍;细胞染色和含量测定表明,核黄素处理后烟草悬浮细胞沉积了更多的木质素,处理24 h和48 h后,木质素含量分别为对照的1.5和1.8倍。【结论】核黄素处理后提高了烟草悬浮细胞PAL、POD活性,积累了更多与抗病有关的次生代谢产物。