维管束特异表达启动子及其顺式调控元件

综述了维管束特异表达启动子及其顺式调控元件和基元序列调控基因表达的研究进展 ,它们在抗细菌、真菌性维管束病害的作物基因工程中具有广阔的应用前景。     

人工合成启动子SAR在拟南芥中的表达分析

利用植物防御基因中的病原诱导响应元件和最小35S启动子(-62～+1),人工合成了启动子SAP,并以GUS基因为报告基因,在转基因拟南芥中分析了合成启动子的表达特性.通过对转基因拟南芥GUS组织染色的分析表明:SAR启动子在子叶、毛刺、根茎交接处和根系中优势表达,在老叶中的表达量高于幼叶,说明SAR启动子具有组织和发育表达特异性.     

一个光合组织特异表达强启动子的分离及功能分析

用接头PCR技术克隆了长度为1415 bp的PNZIP基因启动子, 该启动子具有真核生物启动子的典型特征, 引物延伸实验证明转录起始位点位于翻译起始位点上游122 bp处. 根据PNZIP基因启动子的序列特征, 利用PCR技术对该启动子进行了有目的的缺失, 将5个长度不同的启动子片段分别与报告基因GUS相连接, 构建植物表达载体, 转化烟草. 荧光定量检测结果表明: 5个不同长度的PNZIP启动子均能驱动GUS基因在光合组织中专一表达, 它们的活性随着启动子5′端的逐步缺失而不断地下降; 在叶片组织中长度为1415 bp的PNZIP启动子活性比35S启动子高9倍; PNZIP启动子中存在2个可能与光合组织特异表达有关的新的顺式作用元件, 即GAAATA和GATACT, GATACT元件可能决定基因的光合组织特异性表达, 而GAAATA可能作为增强子提高基因在光合组织的表达强度.     

水稻花药特异启动子Osg6B的序列及功能分析

对水稻花药特异表达基因Ｏｓｇ６启动子简称Ｏｓｇ６Ｂ的全序列进行了测定,结果表明,Ｏｓｇ６Ｂ含有１．７３ｋｂ个核苷酸,与文献报道的该启动子相差仅８个核苷酸,两者核苷酸同源性为９９．５％。将Ｏｓｇ６Ｂ同ＧＵＳ基因编码区相连,将构建成的融合基因用基因抢轰击烟草的花药和幼叶,荧光分析结果为含Ｏｓｇ６Ｂ的ＧＵＳ融合基因在大于２ｍｍ烟草花药中的表达量比对照和幼叶均高出８倍,在小于或等于２ｍｍ的花药中,则高达到     

棉纤维特异基因及其特异启动子的研究进展

棉花是重要经济作物。由于单纯利用常规育种手段很难同步提高棉花产量和品质,利用基因工程与常规育种方法相结合来改良棉花纤维品质是当前品质育种中一个切实可行的方法。本文总结了棉花纤维发育的过程,并对近几年国内外棉纤维发育相关基因及其特异启动子的研究现状进行了综述,以期为利用基因工程手段改良棉花品质提供参考。     

黑芥子酶基因pyk10根特异启动子在番茄中的验证

黑芥子酶是一类催化芥子油苷水解的同工酶,pyk10是一个在拟南芥根和下胚轴中特异表达的黑芥子酶基因.从拟南芥Columbia生态型基因组中克隆的长度为1 450 bp的pykl0基因启动子片段,以gus基因为报告基因构建了植物表达载体pPykG.通过农杆菌介导法,将pykl0的根特异表达启动子以及gus基因转入了番茄中蔬6号,经PCR检测,转化植株中扩增出pyk10启动子特异性条带.组织化学法检测及定量分析,显示pyk10启动子驱动gus基因在番茄的根部特定表达.     

高等植物启动子研究进展

简述了高等植物来源启动子的多种保守顺式调控元件如ＴＡＴＡ盒,转录起始位点,Ｇ盒等,以及双向启动子和可变启动子。着重介绍了受环境包括激素,光,创伤,真菌,逆境等因子诱导表达的植物启动子以及显示出植物发育特异性表达的启动子。     

水稻谷蛋白基因GluC启动子的克隆及表达分析

水稻谷蛋白仅在水稻种子胚乳中表达,其启动子是分离胚乳特异性表达启动子的理想材料。本研究克隆了GluC基因启动子pGluC,生物信息学分析表明pGluC内部含有胚乳特异性表达所需要的Skn-1 motif和ACGT-box元件。将pGluC启动子和7个5'端缺失启动子片段构建到pGPTV-GUS载体上,转化水稻愈伤组织,进行组织化学染色和GUS酶活分析。结果表明：全长及截短的-1 911、-1 611、-1 311 bp启动子均能驱动GUS基因在水稻种子胚乳中高效稳定表达。-999、-451、-203、-102 bp启动子失去了胚乳表达特异性,在根、茎或者叶中也检测到GUS表达。该结果为实现外源目的基因在水稻胚乳中特异高效表达提供了理论依据。     

拟南芥根特异表达基因启动子在烟草中的表达

以拟南芥为材料,利用PCR技术分离pyk10启动子序列,构建了该启动子GUS植物表达载体,农杆菌介导转化烟草,分析该基因在烟草中的表达,以明确拟南芥根特异表达基因pyk10启动子在烟草中的表达特性.结果表明:克隆的pyk10启动子与已报道的pyk10启动子一致性为100%,GUS基因在烟草的根部特异表达,表明该启动子为根部特异表达启动子,为揭示植物根的发生、分化和发育机制,以及培育抗根部病虫害和营养高效利用型转基因烟草奠定了基础.     

植物组织特异性启动子研究

组织特异性启动子可以调控基因在某些特定的器官或组织部位中表达。通过对植物组织特异性启动子进行分类和比较,概述了植物组织特异性启动子的结构特点、功能及研究进展。     

Structure and expression of a root-specific rice gene

Two rice cDNA clones (COS6 and COS9) were isolated, corresponding to genes that were highly expressed in roots from seedlings and mature plants. A genomic clone (GOS9) corresponding to cDNA clone COS9 was isolated and the intron/exon structure was determined by comparing the nucleotide sequences of the mRNA and the genomic clone. 5 ends and 3 ends of the mRNA were determined by primer extension and S1-nuclease mapping respectively. The open reading frame present in GOS9 potentially encodes a protein (14kDa) that does not show any significant homology to other proteins in databases.     

Isolation and characterization of root-specific phosphate transporter promoters from Medicago trunatula

Promoters of phosphate transporter genes MtPT1 and MtPT2 of Medicago truncatula were isolated by utilizing the gene-space sequence information and by screening of a genomic library, respectively. Two reporter genes, beta-glucuronidase (GUS) and green fluorescent protein (GFP) were placed under the control of the MtPT1 and MtPT2 promoters. These chimeric transgenes were introduced into Arabidopsis thaliana and transgenic roots of M. truncatula, and expression patterns of the reporter genes were assayed in plants grown under different phosphate (Pi) concentrations. The expression of GUS and GFP was only observed in root tissues, and the levels of expression decreased with increasing concentrations of Pi. GUS activities in roots of transgenic plants decreased 10-fold when the plants were transferred from 10 microM to 2 mM Pi conditions, however, when the plants were transferred back to 10 microM Pi conditions, GUS expression reversed back to the original level. The two promoters lead to different expression patterns inside root tissues. The MtPT1 promoter leads to preferential expression in root epidermal and cortex cells, while MtPT2 promoter results in strong expression in the vascular cylinder in the center of roots. Promoter deletion analyses revealed possible sequences involved in root specificity and Pi responsiveness. The promoters are valuable tools for defined engineering of plants, particularly for root-specific expression of transgenes.     

Two different novel cis-acting elements of erd1, a clpA homologous Arabidopsis gene function in induction by dehydration stress and dark-induced senescence

Many plant genes have been shown to be induced by water stress and function in stress tolerance. The erd1 gene has been shown to be upregulated in response to both water stress and etiolation. Promoter studies using the erd1 promoter region fused to the luciferase (LUC) reporter gene in Arabidopsis thaliana were performed to identify the putative cis elements involved. Results indicated that the cis elements, responsible for gene expression during dehydration and etiolation, are separately located in two discrete portions of the erd1 promoter. Base substitution analysis showed that a 14-bp region from -599 to -586, and a myc recognition motif from -466 to -461 are necessary for the induction of LUC activity in dehydrated plants. On the other hand, base substitution analysis revealed that both an abscisic acid responsive element (ABRE)-like sequence (from -199 to -195) and an ACGT sequence (from -155 to -152) are required for an etiolation-induced increase in LUC activity. LUC activity measurements from etiolated transgenic plants incubated in either water, N6-benzyleadenine (BA), or a 1% sucrose solution found that while BA was able to delay the increase in LUC activity seen in water-treated plants, no increase in LUC activity was seen in plants incubated in sucrose. These results indicate that the erd1 promoter contains two different regulatory systems that are involved in upregulation by dehydration stress and dark-induced senescence.     

Characterization of a rice gene family encoding root-specific proteins

Two cDNA clones (RCc2 and RCc3) corresponding to mRNAs highly expressed only in root tissues of rice (Oryza sativa L.) seedlings were characterized. Respectively, they encode polypeptides of 146 (14.5 kDa) and 133 amino acids (13.4 kDa) that share high (<70%) sequence similarity with a polypeptide encoded by a cDNA (ZRP3) encoding an mRNA preferentially expressed in young maize roots. Genomic DNA blot analysis revealed that they are members of a small gene family and RCg2, the gene corresponding to RCc2, was isolated. A 1656 bp 5-upstream sequence of RCg2 was translationally fused to a -glucuronidase (GUS) reporter gene and stable introduction of the chimeric construct into rice was confirmed by PCR and genomic DNA blot analyses. Histochemical analysis of transgenic rice plants containing the full-length chimeric gene showed high levels of GUS activity in mature cells and the elongation and maturation zones of primary and secondary roots, and in the root caps, but no GUS activity was detected in root meristematic regions. Surprisingly, high GUS activity was also detected in leaves of the same plants. This raises the possibility that the RCg2 5-upstream element may not be sufficient for the proper spatial control of root specificity in transgenic rice.     

植物非生物胁迫诱导启动子顺式作用元件的研究方法

非生物胁迫严重影响植物生长发育,降低作物产量。植物通过各种途径忍受或抵抗非生物胁迫,主要表现是各种抗非生物胁迫基因的表达。基因表达受其上游启动子及转录因子的调控,目前对抗非生物胁迫诱导启动子顺式作用元件及转录因子的研究成为热点。本文综述了植物非生物胁迫诱导启动子顺式作用元件及转录因子的研究方法,并展望了顺式作用元件及转录因子研究的方向及前景。     

Evaluation in tobacco of the organ specificity and strength of therolD promoter,domain A of the 35S promoter and the 35S2 promoter

In order to study the expression in plants of therolD promoter ofAgrobacterium rhizogenes, we have constructed chimaeric genes placing the coding region of thegusA (uidA) marker gene under control of tworolD promoter fragments of different length. Similar results were obtained with both genes. Expression studies were carried out in transformed R₁ progeny plants. In mature transformed tobacco plants, therolD-gus genes were expressed strongly in roots, and to much lower levels in stems and leaves. This pattern of expression was transmitted to progeny, though the ratio of the level of expression in roots relative to that in leaves was much lower in young seedlings. The degree of root specificity inrolD-gus transformants was less than that of a gene constructed with domain A of the CaMV 35S promoter,domA-gus, but the level of root expression was much higher than with the latter gene. However, the level of expression of therolD-gus genes was less than that of agus gene with a 35S promoter with doubled domain B, 35S²-gus. TherolD-gus genes had a distinctive pattern of expression in roots, compared to that of the two other genes, with the strongest GUS activity observed in the root elongation zone and in vascular tissue, and much less in the root apex.     

谷氨酸棒杆菌人工合成启动子文库的构建及应用

启动子是实现基因精细表达调控的重要工具,广泛应用于微生物的代谢工程改造.谷氨酸棒杆菌是重要的工业底盘,已报道的启动子文库较少且主要是基于完全人工设计的突变序列构建获得.本研究对谷氨酸棒杆菌odhA基因天然启动子的-10区及附近序列进行随机突变,借助rfp报告基因和荧光成像系统进行高通量筛选,构建了包含57个相对强度为2...