一个水稻褐飞虱为害诱导型启动子的克隆

目前组成型启动子在基因工程中的应用最为广泛,然而驱动外源基因在各组织中持续恒定地表达可能引起植物发育迟缓等问题。诱导型启动子能够在特定条件下实现目的基因定时优势表达,最大限度减少由于非必需蛋白在转基因植物内的积累对其造成的伤害。依据基因芯片数据并通过定量RT-PCR验证,找到了一个受水稻褐飞虱(Nilaparavata lugens)为害诱导表达基因(LOC_Os01g73940)。用PCR技术从籼稻品种‘TN1’的基因组中获得Os01g73940上游1 953bp的启动子片段,命名为BPHIP。将BPHIP连接到带有β-glucuronidase(GUS)报告基因的植物表达载体上,通过农杆菌介导转化水稻品种‘中花11’。通过GUS组织化学染色法及定量RT-PCR检测证明,BPHIP是一个受褐飞虱为害和茉莉酸处理诱导上调的启动子。利用此类启动子在基因工程中可以减少对水稻生理方面产生的副作用,对抗虫转基因水稻具有良好的应用价值。     

拟南芥诱导型启动子rd29A的克隆及其功能鉴定

以改善作物抗旱性为目的,采用PCR方法从拟南芥中克隆了诱导型启动子rd29A,序列分析发现克隆的rd29A启动子与已发表的rd29A启动子序列(D13044)的同源性为99.47%。利用DNA重组技术成功构建了rd29A启动子驱动GUS基因的植物表达载体p BI121-rd29-GUS,并通过农杆菌介导法转化烟草,转基因烟草叶片中GUS酶活性的组织化学检测结果表明,rd29A启动子能驱动目的基因的有效表达。因此,可以在后续的马铃薯抗旱转基因研究中直接应用。     

转基因水稻中β-1,3-葡聚糖酶基因启动子缺失体P3的激发子诱导活性

 选择合适的启动子是植物抗病基因工程的关键性因素,病原菌诱导型启动子的获得将为植物提供更多的启动子选择。将大麦β-1,3-葡聚糖酶同工酶GⅢ基因启动子的缺失体片段P3与报告基因gus (β-葡聚糖酸醛苷酶基因)偶联,构建植物表达载体,通过农杆菌介导法转化水稻。PCR结果表明,所获得的10株潮霉素抗性水稻植株均呈PCR阳性;DNA印迹法结果显示,9株含P3/gus的融合基因已整合到水稻基因组DNA中。GUS组织化学染色及荧光法结果显示,P3缺失体驱动的gus在激发子诱导后,获得了高水平表达。T₁代种子的GUS组织化学染色结果也表明,激发子可以诱导高水平的P3活性。     

转基因抗虫水稻研究介绍

(下文续本刊 2 0 0 1年第 2期第 1 8页 )2　转基因程序和方法2 .1　目的基因的构建　　要使一个结构基因发挥作用 ,必须有启动子和终止子的协作。重组 DNA技术以及外源基因的遗传化技术在作物育种中的应用关键因素之一就是如何获得转化体 ,如何使外源基因在受体植物中目标时期的高效表达 ,而植物表达载体的构建是实现上述目标之关键 ,大量实验证明植物基因的转录水平的调控是主要的 ,其中许多转录水平的调控又是通过启动子的顺式作用元件 ( cis- acting elements)和反式作用因子 ( trans- acting factors)的相互作用实现的。因此植物表达…     

含双病原物诱导启动子植物安全表达载体的构建

 本研究用来自烟草的具有高度病原物特异性的两个诱导启动子EAS4和hsr203J,串联驱动GUS基因的表达,同时考虑到转基因植物的安全性,引入双边界序列,构建了含双病原物诱导启动子的植物安全表达载体。将表达载体转化烟草获得转基因植株。分析显示,在正常生长情况下转基因烟草检测不到GUS活性,或活性极低;而受疫霉激发子parasiticein、Phytophthora nicotianea[0]的孢子悬浮液和Ralstonia solanacarum的菌悬液诱导后,转基因烟草叶片中可检测到明显的GUS活性。结果表明,构建的植物表达载体所含双病原物诱导启动子均具有良好的诱导活性,可用于植物遗传转化。     

田旋花 EPSPS 基因启动子的克隆、序列分析及转化

根据已克隆的田旋花（ Convolvulus arvensis L．） EPSPS基因mRNA序列设计引物,通过染色体步移技术获得了1142 bp的EPSPS-P启动子片段（ GenBank 登录号：KC107822）。对启动子序列分析显示,该片段富含A/T碱基、TATA-box、CAAT-box及其他作用元件如GATA-motif、TC-rich repeats、sp1等。将该启动子与GUS报告基因连接以构建植物表达载体,利用农杆菌介导法获得转基因拟南芥;PCR和GUS组织化学染色分析证明, EPSPS-P已转化到拟南芥中。     

病原细菌无毒基因avrD介导的抗赤星病转基因烟草

 利用从病原细菌Pseudomonas syringae pv.tomato中获得的无毒基因avrD片段与病原菌诱导型的特异启动子PⅠⅡ、CHS和PAL构建成表达盒,通过土壤农杆菌分别转化烟草品种K326和Bottom Special。经烟草赤星病菌孢子定量接种转基因植株及其离体叶片,筛选到一批高抗植株。Southern分子杂交检测证明,启动子-avrD嵌合基因已整合到寄主染色体并获得表达。     

芜菁WRKY group Ⅲ家族鉴定及其对根肿病菌和激素的响应

WRKY转录因子是植物中特有的一类转录因子，已有研究表明WRKY groupⅢ家族众多基因在抵御生物胁迫的过程中发挥着重要的作用。为了解WRKY groupⅢ家族成员在芜菁根肿病抗性中的潜在功能，利用生物信息学分析方法，鉴定到21个候选WRKY groupⅢ家族基因，其不均匀分布于芜菁的9条染色体，编码蛋白包含258～916个不等的氨基酸残基，含0～8个不等的motif结构，大部分成员包含按顺序排列的motif 4-5-7/(8-2)-1-3。WRKY groupⅢ家族各成员启动子区域含有大量光、激素和逆境胁迫响应元件，在激素响应元件中以Me-JA最多。qRT-PCR结果显示WRKY groupⅢ家族基因可响应根肿病菌诱导，其中BrWRKY46-2、BrWRKY70-2在抗病材料根部表达量显著上调，感病材料中降低；经外源SA诱导后表达量上调，Me-JA诱导后下调，表现出相反的表达模式。亚细胞定位结果显示，BrWRKY46-2和BrWRKY70-2均定位于细胞核中。推测芜菁WRKY groupⅢ家族基因广泛参与了根肿菌响应过程，且BrWRKY46-2和BrWRKY70-2与芜菁品种抗病和...     

GUS基因插入导致的小麦条锈菌突变体遗传稳定性和毒性变异研究

 对3个通过基因枪转化GUS基因获得的小麦条锈菌毒性突变体,进行GUS基因的表达活性检测、插入片段DNA的PCR电泳检测和Southern杂交验证;同时用17个国内鉴别寄主和37个黄淮麦区小麦主栽品种对这3个突变菌株进行了毒性谱的测定。结果表明突变菌株的外源GUS基因可稳定遗传;插入的外源基因片段引起了菌株毒性谱的改变。外源基因的插入位点与条锈菌的毒性基因相关。研究还表明插入突变体具有相对的遗传稳定性。研究结果可为克隆小麦条锈菌毒性基因和探明毒性基因的作用机理奠定理论基础。     

大豆中疫霉诱导性GmDRRP基因启动子的克隆与功能分析

本研究根据大豆与疫霉互作的基因芯片数据,筛选了一个受疫霉诱导表达的大豆抗病相关基因(Gm DRRP,glycine max disease resistance response protein),并对其启动子响应疫霉侵染的功能进行了研究。序列分析表明该基因编码一个大豆抗病相关蛋白。分别用SA、Me JA、ABA、ETH和GA3五种激素处理后,发现该基因的表达受激素的抑制。克隆其转录起始位点上游1 590 bp的启动子区,生物信息学分析发现该区域包含多个已知与逆境响应相关的顺式元件。进一步将启动子构建在融合Gus报告基因的植物表达载体上,分别瞬时转化烟草和稳定转化大豆根毛,并检测了Gus报告基因的表达情况。结果表明,Gm DRRP启动子均能在两种体系中不同程度的受疫霉诱导,其表达模式为接种后0.5 h被快速诱导,并在2 h时显著提高。根据生物信息学对顺式元件的预测结果,对启动子进行了分段分析,获得了一个222 bp的小片段,其疫霉诱导表达能力是全长启动子的34%。以上结果表明大豆的Gm DRRP启动子能被疫霉快速诱导。     

Analysis of the expression patterns of the Arabidopsis thaliana tubulin-1 and Zea mays ubiquitin-1 promoters in rice plants in association with nematode infection

The temporal and spatial expression of three promoters was investigated in transgenic rice plants using promoter-β-glucuronidase (gusA) reporter gene fusions. The promoters studied were ubiquitin-1 (UBI-1) of Zea mays, Cauliflower Mosaic Virus 35S gene (CaMV35S) and a tubulin gene (TUB-1) of Arabidopsis thaliana. The TUB-1 promoter provided 7.32-fold more GUS activity in roots relative to tillers. This was significantly different from the corresponding value of 2.82-fold for CaMV35S but not from that of 4.55-fold for UBI-1, activity of both promoters was higher in the root tips and zone of elongation than mature roots. This younger root tissue represented a declining proportion of the expanding root system with time. Older tissue expressing GUS under control of the TUB-1 promoter showed a steeper decline in activity with time than occurred with the UBI-1 promoter. Nematode infection did not alter the overall pattern of expression from the two promoters, except that the giant cells induced by Meloidogyne incognita retained TUB-1 promoter activity as roots matured. Pratylenchus zeae invaded older root regions than M. incognita and no changes in promoter activity were detected where it fed. The results suggest the TUB-1 promoter has characteristics that favour its use for delivering anti-feedants, such as cysteine proteinase inhibitors, to M. incognita.     

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。     

A Promoter from Pea Gene DRR206 Is Suitable to Regulate an Elicitor-Coding Gene and Develop Disease Resistance

ABSTRACT Plant nonhost disease resistance is characterized by the induction of multiple defense genes. The pea DRR206 gene is induced following inoculation with pathogens and treatment with abiotic agents, and moderately induced by wounding. A deletion series of DRR206 promoter segments was fused with the beta-glucuronidase (GUS) reporter gene and transiently transferred to tobacco, potato, and pea. GUS activity revealed that two upstream regions of the DRR206 promoter were particularly important for activation in the three plant species. Putative cis regulatory elements within the DRR206 promoter included a wound/pathogen- inducible box (W/P-box) and a WRKY box (W-box). Gel shift assays with nuclear extracts from treated and untreated tissue with the W/P-box revealed both similar and unique protein-DNA complexes from pea, potato, and tobacco. Tobacco was stably transformed with gene constructs of the DRR206 promoter fused with a DNase elicitor gene from Fusarium solani f. sp. phaseoli, FsphDNase. Pathogenicity tests indicated that the FsphDNase elicitor conferred resistance against Pseudomonas syringae pv. tabaci and Alternaria alternata in tobacco. Transgenic potatoes showed some sensitivity to the FsphDNase gene providing less protection against Phytophthora infestans. Thus, the elicitor-coding gene, FsphDNase, is capable of generating resistance in a heterologous plant system (tobacco) when fused with defined regions of the pea DRR206 promoter.     

Expression of Oryzacystatin I and II in Alfalfa Increases Resistance to the Root-Lesion Nematode

ABSTRACT Digestive cysteine proteinases have been isolated from plant-parasitic nematodes as well as coleopteran and hemipteran insects. Phytocystatins, inhibitors of cysteine proteinases, are found in a number of plants where they may play a role in defense against pathogens and pests. The cDNAs of the phytocystatins from rice, oryzacystatin I (OC-I) and oryzacystatin II (OC-II), were expressed in alfalfa (Medicago sativa) plants under the control of the potato protease inhibitor II (PinII) promoter and the plants were evaluated for resistance to the root-lesion nematode (Pratylenchus penetrans). A PinII-beta-glucuronidase (GUS) gene was introduced into alfalfa to determine the pattern of gene expression from this promoter. Constitutive GUS expression was observed in leaf and root vascular tissue, and in some plants, expression was observed in leaf mesophyll cells. Mechanical wounding of leaves increased GUS expression approximately twofold over 24 h. Inoculation with root-lesion nematodes resulted in localized GUS expression. Populations of root-lesion nematodes in alfalfa roots from one line containing the PinII::OC-I transgene and one line containing the PinII::OC-II transgene were reduced 29 and 32%, respectively, compared with a transgenic control line. These results suggest that oryzacystatins have the potential to confer increased resistance to the root-lesion nematode in alfalfa.     

水稻抗病基因Xa21转入3个粳稻品种的抗性初步分析

 通过农杆菌介导转化,将已克隆的水稻白叶枯病抗性基因Xa21转入我国辽宁省3个粳稻品种:沈农606、辽粳454、辽粳294,共获得独立转基因系205个。通过PCR、GUS染色和Southern blot分析,证明Xa21基因已经整合到3个粳稻栽培品种的基因组中。通过温室接种菲律宾白叶枯病小种6的代表菌系PXO99,T₀代转基因水稻除了3-34表现部分抗性外,其它的转基因材料均表现高度抗病,表明Xa21转基因水稻已经获得抗性;温室接种T₁和T₂代试验表明,单拷贝整合的转化体呈现3:1分离,同时单拷贝3-34材料也表现部分抗性和感病3:1分离,证明已整合的Xa21基因能稳定遗传;同时对3-34部分抗性机制进行了分子生物学检测,证明GUS基因全部丢失、Xa21基因部分丢失并导致部分抗性。获得部分抗性材料,对于深入研究Xa21基因的功能区和研究抗病分子机制具有重要的意义。     

Synthetic tetramer of a Phytophthora sojae-inducible fragment from soybean GmaSKTI36 promoter improves its pathogen induction activities

Using pathogen-induced promoters to control expression of the functional genes in transgenic plants may greatly increase the chances of boosting disease resistance. However, the number of the inducible promoters is limited. Here, we found that soybean GmaSKTI36 gene is strongly induced upon Phytophthora sojae infection. Functional analysis showed that its promoter could mediate rapid and strong induction of GUS expression upon pathogen infection in both Nicotiana benthamiana leaves and soybean hairy roots. Then, a 122 bp fragment that was critical to the activity was successfully identified by a progressive 5′ deletion analysis. Importantly, we found that a synthetic promoter by tetramerizing this fragment could confer strong P. sojae induction activities. Overall, the results suggested that the GmaSKTI36 promoter, the 122 bp fragment, and the synthetic promoter are potentially useful pathogen-inducible promoters.