A wheat histone H3 promoter confers cell division-dependent and -independent expression of the gus A gene in transgenic rice plants

To investigate developmental regulation of wheat histone H3 gene expression, the H3 promoter, which has its upstream sequence to ?1711 (relative to the cap site as +1), was fused to the coding region of the gus A gene (?1711H3/GUS) and introduced into a monocot plant, rice. Detailed histochemical analysis revealed two distinct types of GUS expression in transgenic rice plants; one is cell division-dependent found in the apical meristem of shoots and roots and in young leaves, and another is cell division-independent detected in flower tissues including the anther wall and the pistil. In this study, replication-dependent expression occurring in non-dividing cells which undergo endoreduplication could not be discriminated from strict replication-independent expression. The observed expression pattern in different parts of roots suggested that the level of the H3/GUS gene expression is well correlated with activity of cell division in roots. To identify 5′ sequences of the H3 promoter necessary for an accurate regulation of the GUS expression, two constructs containing truncated promoters, ?908H3/GUS and ?185H3/GUS, were analyzed in transiently expressed protoplasts, stably transformed calli and transgenic plants. The results indicated that the region from ?909 to ?1711 contains the positive cis-acting element(s) and that the proximal promoter region (up to ?185) containing the conserved hexamer, octamer and nonamer motifs is sufficient to direct both cell division-dependent and -independent expression. The use of the meristem of roots regenerated from transformed calli for the analysis of cell division-dependent expression of plant genes is discussed.     

A strong constitutive gene expression system derived from <Emphasis Type="Italic">ibAGP1</Emphasis> promoter and its transit peptide

To develop a strong constitutive gene expression system, the activities of ibAGP1 promoter and its transit peptide were investigated using transgenic Arabidopsis and a GUS reporter gene. The ibAGP1 promoter directed GUS expression in almost entire tissues including rosette leaf, inflorescence stem, inflorescence, cauline leaf and root, suggesting that the ibAGP1 promoter is a constitutive promoter. GUS expression mediated by ibAGP1 promoter was weaker than that by CaMV35S promoter in all tissue types, but when GUS protein was targeted to plastids with the aid of the ibAGP1 transit peptide, GUS levels increased to higher levels in lamina, petiole and cauline leaf compared to those produced by CaMV35S promoter. The enhancing effect of ibAGP1 transit peptide on the accumulation of foreign protein was tissue-specific; accumulation was high in lamina and inflorescence, but low in root and primary inflorescence stem. The transit peptide effect in the leaves was maintained highly regardless of developmental stages of plants. The ibAGP1 promoter and its transit peptide also directed strong GUS gene expression in transiently expressed tobacco leaves. These results suggest that the ibAGP1 promoter and its transit peptide are a strong constitutive foreign gene expression system for transgenesis of dicot plants.     

獐茅HAK基因表达调控区的分离及其在水稻中的功能分析

獐茅高亲和性K+转运蛋白基因(AlHAK1)是从单子叶禾本科盐生植物獐茅(Aeluropus littoralis(Gouan)Parl)中克隆,对于细胞营养和离子渗透调节起关键作用。为了进一步了解AlHAK1基因的表达调控机制,文章采用基因组步移法分离了AlHAK1基因转录起始位点上游长度约1.3 kb的启动子区域。启动子顺式元件分析显示该序列具有典型的TATA和CAAT盒,以及一些与植物生长发育和环境响应相关的顺式元件。为了明确AlHAK1启动子的功能,将其与GUS基因融合构建到植物表达载体pCAMBIA1301上,通过农杆菌介导转化法导入水稻中。对转基因植株进行GUS组织化学染色,结果显示在转化AlHAK1启动子水稻的根、茎、叶、花药和内外稃部位均检测到GUS活性。GUS荧光定量分析显示AlHAK1启动子调节GUS表达活性低于组成型启动子CaMV35S和Ubiquitin,但其根部和茎部的GUS活性相对较高。对转化植株进行不同胁迫处理后检测GUS活性,结果表明受到ABA、干旱、高温的诱导后其茎部和根部GUS活性有所提高,推测位于该启动子-682 bp的HSE元件和-1 268 bp的MybBS元件可能在高温、ABA和干旱诱导的表达调控中起作用。     

Two novel positive cis-regulatory elements involved in green tissue-specific promoter activity in rice (Oryza sativa L ssp.)

In plant genetic engineering, using tissue-specific promoters to control the expression of target gene is an effective way to avoid potential negative effects of using constitutive promoter, such as metabolic burden and so on. However, until now, there are few tissue-specific promoters with strong and reliable expression that could be used in crop biotechnology application. In this study, based on microarray and RT-PCR data, we identified a rice green tissue-specific expression gene DX1 (LOC_Os12g33120). The expression pattern of DX1 gene promoter was examined by using the β-glucuronidase (GUS) reporter gene and analyzed in transgenic rice plants in different tissues. Histochemical assays and quantitative analyses of GUS activity confirmed that P (DX1):GUS was highly expressed in green tissues. To identify the regulatory elements controlling the expression of the DX1 gene, a series of 5' and 3' deletions of DX1 promoter were fused to GUS gene and stably introduced into rice plants. In addition, gel mobility shift assays and site-directed mutagenesis studies were used, allowing for the identification of two novel tissue-specific cis-acting elements (GSE1 and GSE2) within P(DX1). GSE1 acted as a positive regulator in all green tissues (leaf, sheath, stem and panicle). Compared with GSE1, GSE2 acted as a positive regulator only in sheath and stem tissue, and had a weaker effect on gene expression. In addition, P(DX1):GUS was not expressed in anther and seed, this characteristic reduced the potential ecological risk and potential food safety issues. Taken together, our results strongly suggest that the identified promoter, P(DX1), and its cis regulatory elements, GSE1 and GSE2, are potentially useful in the field of rice transgenic breeding. KEY MESSAGE: We have isolated and characterized the rice green tissue-specific promoter P(DX1), and identified two novel positive cis-acting elements in P(DX1).     

竹节花黄斑驳病毒启动子的缺失分析及功能

竹节花黄斑驳病毒(CoYMV)是侵染单子叶植物竹节花的一 种双链环状DNA病毒,它的启动子可介导外源基因在烟草韧皮部特异表达。为了研究其组织 特异性表达的最佳启动子区域,对CoYMV启动子进行了5′端五种不同长度的缺失分析,用不同长度的启动子片段与GUS基因及NOS3′端转录中止序列构建了全长启动子及5 个缺失启动子序列的六个嵌合GUS基因植物表达载体。利用农杆菌将上述嵌合基因转化烟草 外植体后,每种表达载体都获得了一批转基因烟草植株。转化再生烟草植株的PCR分析、GUS 酶活测定及GUS组织染色的结果表明六种类型的嵌合基因已整合到烟草染色体中,并有五种 表达出GUS活性。缺失到870bp的启动子比全长启动子(1040bp)的活性约高78%,870bp比585bp启动子介导的GUS活性略高但差别不明显,缺失到447和232时GUS活性有明显下 降,但仍具有韧皮部特异表达的特性。当缺失到TATA box附近的44bp时启动子丧失组织特 异性,GUS活性也降低到测不出来的水平。以上结果表明CoYMV启动子从转录起始位点上游 870bp～230bp及232bp下游区分别与启动子的活性和韧皮部组织特异性密切相关,870bp上游可能存在一个负调控序列,所以该启动子的活性和组织特异性的最佳调控区应在87 0bp或585bp的下游区。CoYMV启动子与35S启动子驱动GUS基因在烟草中表达的活性相比, 前者为后者的70%左右,考虑到前者仅在韧皮部细胞表达而后者为组成型表达,所以CoYMV启 动子在韧皮部的活性可能与35S启动子相当或更高。CoYMV启动子在其它转基因植物中驱动外 源基因表达的特点正在研究中。     

Intragenic control of expression of a rice MADS box gene OsMADS1

OsMADS1 is a rice MADS box gene necessary for floral development. To identify the key cis-regulatory regions for its expression, we utilized transgenic rice plants expressing GUS fusion constructs. Histochemical analysis revealed that the 5.7-kb OsMADS1 intragenic sequences, encompassing exon 1, intron 1, and a part of exon 2, together with the 1.9-kb 5' upstream promoter region, are required for the GUS expression pattern that coincides with flower-preferential expression of OsMADS1. In contrast, the 5' upstream promoter sequence lacking this intragenic region caused ectopic expression of the reporter gene in both vegetative and reproductive tissues. Notably, incorporation of the intragenic region into the CaMV35S promoter directed the GUS expression pattern similar to that of the endogenous spatial expression of OsMADS1 in flowers. In addition, our transient gene expression assay revealed that the large first intron following the CaMV35S minimal promoter enhances flower-preferential expression of GUS. These results suggest that the OsMADS1 intragenic sequence, largely intron 1, contains a key regulatory region(s) essential for expression.     

Chromosomal location plays a role in regulation of aflatoxin gene expression in Aspergillus parasiticus.

The nor-1 gene in the filamentous fungus Aspergillus parasiticus encodes a ketoreductase involved in aflatoxin biosynthesis. To study environmental influences on nor-1 expression, we generated plasmid pAPGUSNNB containing a nor-1 promoter-beta-glucuronidase (GUS) (encoded by uidA) reporter fusion with niaD (encodes nitrate reductase) as a selectable marker. niaD transformants of A. parasiticus strain NR-1 (niaD) carried pAPGUSNNB integrated predominantly at the nor-1 or niaD locus. Expression of the native nor-1 and nor-1::GUS reporter was compared in transformants grown under aflatoxin-inducing conditions by Northern and Western analyses and by qualitative and quantitative GUS activity assays. The timing and level of nor-1 promoter function with pAPGUSNNB integrated at nor-1 was similar to that observed for the native nor-1 gene. In contrast, nor-1 promoter activity in pAPGUSNNB and a second nor-1::GUS reporter construct, pBNG3.0, was not detectable when integration occurred at niaD. Because niaD-dependent regulation could account for the absence of expression at niaD, a third chromosomal location was analyzed using pAPGUSNP, which contained nor-1::GUS plus pyrG (encodes OMP decarboxylase) as a selectable marker. GUS expression was detectable only when pAPGUSNP integrated at nor-1 and was not detectable at pyrG, even under growth conditions that required pyrG expression. nor-1::GUS is regulated similarly to the native nor-1 gene when it is integrated at its homologous site within the aflatoxin gene cluster but is not expressed at native nor-1 levels at two locations outside of the aflatoxin gene cluster. We conclude that the GUS reporter system can be used effectively to measure nor-1 promoter activity and that nor-1 is subject to position-dependent regulation in the A. parasiticus chromosome.     

AtSTP3启动子控制的外源基因在转基因水稻中的特异表达研究

利用PCR技术从哥伦比亚型拟南芥基因组DNA中分离了AtSTP3绿色组织特异表达的启动子,序列分析表明,扩增片段(1774bp)与已报道序列的相应区域同源性达99.9%。将其与GUS报告基因融合在一起,构建了植物表达载体,并由农杆菌介导法导入水稻品种‘中花11’中。对转基因水稻植株中的GUS活性进行定性与定量测定结果表明,AtSTP3启动子可驱动GUS报告基因在转基因水稻植株叶片中特异性表达,而在根和种子等器官中不表达或表达活性极弱,AtSTP3启动子表现出明显的组织特异性。     

Transformation of peanut with a soybean vspB promoter‐uidA chimeric gene. I. Optimization of a transformation system and analysis of GUS expression in primary transgenic tissues and plants

Direct DNA delivery via microprojectile bombardment has become an established approach for gene transfer into peanut ( Arachis hypogaea L.). To optimize our transformation protocol and to simultaneously explore the function of a heterologous promoter whose activity is developmentally regulated, embryogenic cultures from three peanut cultivars were bombarded with two plasmid constructs containing a uidA gene controlled by either a soybean vegetative storage protein gene promoter or a cauliflower mosaic virus 35S promoter. We found that GUS transient expression was useful to predict stable transformation and confirmed that image analysis could provide a quick and efficient method for semi‐quantitation of transient expression. One hundred and sixty hygromycin‐resistant cell lines were recovered from and maintained on selective medium, and those tested by Southern blot analysis showed integration of the foreign gene. Over 200 transgenic plants were regenerated from 38 cell lines. More than 100 plants from 32 cell lines flowered and 79 plants from 19 cell lines produced pods. Over 1000 R₁ seeds were harvested. Analysis of expression in primary transgenic plants showed that GUS expression driven by the vspB promoter was modulated by chemical and positional information.     

Developmental regulation of peach ACC oxidase promoter--GUS fusions in transgenic tomato fruits

A genomic DNA sequence (PpACO1) encoding 1-aminocyclopropane-1-carboxylic acid oxidase (ACO) from peach (Prunus persica L. Batsch cv. Loring) was isolated. It has four exons interrupted by three introns and 2.9 kb of flanking region 5' of the translational start codon. Previous work with the cDNA demonstrated that accumulation of the peach ACO message correlated with increasing amounts of ethylene synthesized by the fruit as they ripened. To identify regulatory elements in the peach ACC oxidase gene, chimeric fusions between 403, 610, 901, 1319, 2141, and 2919 bp of the 5' flanking region of the PpACO1 sequence and the beta-glucuronidase (GUS) coding sequence were constructed and used to transform tomato (Lycopersicon esculentum [Mill] cv. Pixie). Fruits from the various promoter lines were analysed for GUS expression by histochemical GUS staining, GUS quantitative enzyme activity determination, and measuring the relative amounts of GUS mRNA. Constructs with the smallest promoter of 403 bp had significant GUS expression in fruit, but not in other tissues, indicating the presence of a region that affects tissue-specific expression. An increase in GUS expression was observed with promoters longer than 901 bp, indicating an enhancer region between -1319 and -901. The full-length promoter of 2919 bp directed GUS expression in the green stage of fruit development, and increased GUS expression as fruit matured, indicating a regulatory region between -2919 and -2141 that controls the temporal expression of the gene in fruit. Only the full-length promoter sequence demonstrated responsiveness to ethylene.     

根癌农杆菌介导的苜蓿体胚转化

以苜蓿体细胞胚胎作为根癌农杆菌介导转化的受体,通过对GUS基因瞬时表达率的分析,研究该转化体系的最佳实验参数。实验结果显示,负压处理10min和共培养5d时表达率最高(可达17．4％)。以这一转化方法分别对带有3种不同启动于的表达载体进行比较,发现由CMV35S启动于驱动的GUS基因的瞬时表达率可达82．7％,Ubil启动于驱动的可达57．8％,而Actl启动于驱动的则未见表达。