番茄根特异表达基因LeGRP2启动子的克隆及其在拟南芥的表达分析

目的克隆获得番茄根特异表达启动子,为利用基因工程技术创制番茄新种质奠定基础。方法利用Clontech公司的基因组步移(genome walking)技术,扩增番茄根特异表达基因LeGRP2的上游调控序列,并构建植物表达载体,利用农杆菌介导法转化拟南芥,以GUS为报告基因研究该调控序列的组织表达特异性。结果以番茄基因组DNA为模板,经过2次基因组步移,获得了LeGRP2基因上游1959bp的调控序列(GenBank登录号:EU262719),分析发现含有9个与根特异表达相关的顺式作用元件ROOTMOTIFTAPOX1。转基因拟南芥的组织化学染色分析表明,GUS基因主要在拟南芥的根部特异表达。结论克隆获得了番茄LeGRP2基因启动子,该启动子主要在转基因拟南芥根部表达GUS基因,具有较强的根表达特异性。     

利用5'LongSAGE标签分析蜜蜂肌球蛋白调节性轻链基因MLC-2选择性转录起始位点

使用西方蜜蜂雄蜂头部5’LongSAGE文库中的肌球蛋白调节性轻链基因MLC-2的标签序列,在蜜蜂全基因组序列上定位了该基因的转录起始位点(TSS),并进而预测了其启动子的结构。结果显示:蜜蜂MLC-2基因存在17个TSS,其中16个定位于MLC-2基因开放阅读框上游100bp的范围内。MLC-2基因的各TSS的使用效率不同,其中有3个优势TSS,由之起始的转录本分别占该基因总转录本数量的9.76%、54.47%和11.38%。从碱基组成来看,蜜蜂MLC-2基因TSS的第1个碱基为A、G、T、C的概率分别为58.8%、29.4%、0和11.8%。MLC-2基因的启动子结构预测结果表明,在TSS上游的300bp区域内有3个核心启动子元件。研究结果对研究蜜蜂MLC-2基因的表达调控与确定其全长cDNA序列具有重要意义。     

观赏海棠不同品种MYB10启动子结构分析

[目的]观赏海棠叶片色泽类型多样,为了研究McMYB10基因对不同叶色观赏海棠品种呈色的影响.[方法]利用PCR克隆的方法,从两个极端叶色观赏海棠品种中克隆得到两个McMYB10启动子,同时对这两个启动子在5个不同叶色类型的观赏海棠分布进行检测.[结果]McMYB10启动子在不同观赏海棠品种中存在两种不同的类型,即R1型和R6型;在McMYB10启动子中存在逆境、激素、光等多种顺式作用元件,同时在变色类观赏海棠品种中,R1和R6型启动子都存在.[结论]R1和R6型启动子在不同叶色的观赏海棠品种中,可能对叶片的呈色起较为重要的作用.     

花器官特异启动子的研究进展

简单介绍了植物花器官特异性启动子的克隆、顺式表达元件及在改良花形、花色、花香等方面的研究应用。     

半滑舌鳎vasa调控区的克隆、表达载体构建及其驱动活性检测

为研究半滑舌鳎(Cynoglossus semilaevis)vasa(Csvasa)基因调控区的功能,在已克隆的Csvasa基因编码序列的基础上,采用基因组步移和PCR扩增的方法克隆得到Csvasa调控区,通过生物信息学方法分析vasa基因5′区,并构建了含Csvasa基因调控区的绿色荧光蛋白(GFP)表达载体(p Csvasa-GFP-T),进一步通过显微注射技术初步验证调控区的驱动活性。结果表明,通过基因组步移和PCR扩增获得Csvasa 5′区5 166 bp和3′区1 655 bp,利用在线生物信息学软件对5′区序列进行分析,发现在转录起始点上游26 bp处存在保守的TATA框,以及潜在的转录因子结合点如SRY、Oct-1、Sox-5、CREB、GATA、AP-1、C/EBP、Sp-1、c-Myc、HNF、NKX2-5、V-Myb等。通过显微注射技术,将所构建的p Csvasa-GFP-T表达载体注射于青鳉(Oryzias latipes)受精卵并进行培养观测,发现Csvasa调控区能够驱动GFP在青鳉胚胎内表达,荧光表达率为81%。将有荧光的胚胎培养为成鱼,检测外源基因的整合率为11.5%。这些结果为进一步研究半滑舌鳎原始生殖细胞(PGCs)的标记、追踪和操作研究以及半滑舌鳎的性别控制等奠定了基础。     

大口黑鲈GHRH基因启动子区域序列分析及其活性检测

生长激素释放激素(growth hormone releasing hormone,GHRH)是下丘脑弓状核合成和分泌的小分子多肽,其主要功能是调节垂体细胞合成和释放生长激素。为研究大口黑鲈(Micropterus salmoides)GHRH基因5’侧翼启动子区域的活性和该区域中潜在的转录因子对GHRH基因表达的调控作用,对该基因5’端启动子区域约1400 bp长度的片段进行序列分析,预测顺式作用元件,获得了Oct-1、SP1、NF-1、C/EBPalp和C/EBP等多个潜在的调控GHRH基因表达的调节因子结合位点序列。在包括外显子1和内含子1的GHRH基因5’侧翼区两侧加入两个限制性酶切位点Xho I和Bam H I,对其进行改造,并将该片段插入红色荧光蛋白报告基因载体p DsRed2-1,构建了重组表达质粒pGHRH1-RFP。同时,用不含有外显子1和内含子1的GHRH基因5’侧翼区构建重组表达质粒p GHRH-RFP。将质粒pGHRH1-RFP和p GHRH-RFP转染鲤(Cyprinus carpio)上皮细胞(epithelioma papillosum cyprinid,EPC)。经过48 h的培养,在pGHRH1-RFP转染的部分细胞中检测到红色荧光蛋白表达。又将pGHRH1-RFP或p GHRH-RFP质粒注射到斑马鱼(Danio rerio)一细胞或二细胞期的胚胎中,注射了pGHRH1-RFP的胚胎在受精后48 h约有22.5%能检测到有红色荧光蛋白表达,受精后72 h约有29%的仔鱼检测到红色荧光蛋白表达。实验结果表明,目前分离到的GHRH基因5’侧翼序列具有启动基因表达的活性,且该基因的内含子1和外显子1是启动子的活性所必需的。另外,pGHRH1-RFP质粒注射的斑马鱼胚胎只能在胚胎和仔鱼的脊椎和肌肉中检测到RFP的表达,而在脑中没有检测到表达。推测扩增到的大口黑鲈GHRH启动子序列1407 bp(-1043 bp~362 bp)只是起到了驱动RFP脊椎和骨骼肌表达的作用,而不包括驱动在脑组织中特异性表达的启动子,本研究为GHRH基因功能的深入分析奠定了基础。     

灰树花gpd-GF启动子的克隆与表达载体的构建

根据已经报道有强启动子活性的香菇gpd启动子设计引物,从灰树花基因组PCR扩增获得大小分别为1018,615bp的2个片段gpd-GF1和gpd-GF2,通过DNA序列测定得知二者的大小分别为1018,615bp,经NCB1中的BLASTN比较,gpd-GF1,gpd-GF2与已报道的香菇中克隆的gLeGPD基因上游序列的同源性分别为96%,98%,通过启动子预测软件分析,结果表明,gpd-GF1和gpd-GF2含有多个顺式作用元件如TATA box,GAtA box,CAAT box等,初步证实 gpd-GF1,gpd-GF2可能有较强的启动子活性。将gpd-GF1,gpd-GF2分别与切除CaMV35S启动子的pCAMB1A1301大片段进行亚克隆,构建成表达载体pCBgpdGF1和pCBgodGF2。     

The Exploitation of Nematode-Responsive Plant Genes in Novel Nematode Control Methods

The molecular interactions between plants and sedentary nematodes are undergoing intense study, not only for reasons of fundamental research but also for the potential benefits to agriculture. The present technology allows the transformation of an increasing number of crop plants, providing new ways to introduce resistance against plant-parasitic nematodes. The ability of sedentary nematodes to induce specialized feeding sites in plant roots is one of the most fascinating aspects of this host–parasite interaction. Molecular approaches have been initiated to identify and characterize plant genes altered in expression after infection by sedentary nematodes. The results obtained indicate that many genes indeed become up-regulated upon nematode infection. Surprisingly, several so-called constitutive promoters that are normally used to achieve high expression in plant cells are completely ‘silenced’ in the feeding sites within days after nematode infection. Generally, there are two options available for the genetic engineering of nematode resistance: the synthesis of anti-nematode proteins or the localized production of a cytotoxic protein that interferes with the development of feeding cells. Nematode-induced promoters are very useful for the production by plants of sufficiently high levels of anti-nematode proteins at feeding sites. Alternatively, interfering with feeding-cell development is somewhat similar to the hypersensitive response evoked by nematodes in a naturally resistant plant. Here, destruction of specific plant cells can be achieved by the localized expression of a cytotoxin such as barnase, a potent ribonuclease. This approach, however, calls for a highly specific ‘non-leaky’ promoter, which is active only in the feeding cells. Another possibility is to use a two-component system, where the leakiness of the promoter in other tissues is counterbalanced by the constitutive expression of a neutralizing gene.     

Conditional expression of harpinPsscauses yeast cell death that shares features of cell death pathway with harpinPss-mediated plant hypersensitive response (HR)

Conditional expression of harpin_Psscauses yeast cell death that shares features of cell death pathway with harpin_Pss-mediated plant hypersensitive response (HR).Pseudomonas syringae pv.syringae 61 hrp Z gene encodes harpin_Pss, a 34.7 kD extracellular protein that elicits a hypersensitive response (HR) in plants. Conditional expression of either full-length or truncated hrp Z sequences under the GAL1 promoter caused cell death in Saccharomyces cerevisiae Y187. Plating of pYEUT- hrp Z transformants on a medium containing galactose resulted in complete inhibition of colony formation, whereas their growth on a glucose-based medium was unaffected. Western blot analysis confirmed the expression of harpin_Pssin yeast cells transformed with pYEUT- hrp Z and grown in galactose-containing medium. A time-dependent decline in the percentage of trypan blue-excluding cells in cultures of pYEUT- hrp Z transformants was observed when cultured on galactose-containing medium. Similarly, the number of viable cells reduced to about 50% within 6 h. There were similarities in the harpin_Pss-mediated cell death in plants and yeast cell death (YCD). Galactose-induced cell death in pYEUT-hrp Z transformants of S. cerevisiae Y187 was suppressed by a protein kinase inhibitor K252a (10 μ M). The viability of pYEUT- hrp Z transformants was prolonged in the presence of 100 U ml⁻¹catalase suggesting a role for the oxidative burst in YCD that was further supported by the flow cytometric patterns of propidium iodide uptake by yeast cells. Overall, it appears that yeast provides a useful model system to understand the molecular mechanism of harpin_Pss-mediated cell death.     

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.