Expression ofArabidopsis tryptophan biosynthetic pathway genes: effect of the 5′ coding region of phosphoribosylanthranilate isomerase gene

There are three non-allelic isogenes encoding phosphoribosylanthranilate isomerase (PAI) inArabidopsis thaliana. The expression plasmids were constructed by fusion of the GUS reporter gene to the three PAI promoters with or without the 5′ region encoding PAI N-terminal polypeptides and transferred into Arabidopsis plants byAgrobacterium tumefaciens. Analysis of GUS activity revealed that the PAI 5′ coding region was necessary for high expression of GUS activity. GUS activity in transgenic plants transformed with the expression plasmids containing the 5′ coding region of PAH or PAI3 was 60–100-fold higher than that without the corresponding 5′ region. However, the effect of 5’ coding region of PAI2 gene on the GUS activity was very small (only about 1 time difference). The GUS histochemical staining showed a similar result as revealed by GUS activity assay. It was expressed in the mesophyll cells and guard cells, but not in the epidermic cells, indicating that the N-terminal polypeptides encoded by the 5′ region of PAI genes have the function of PTP.     

Arabidopsis phosphoribosylanthranilate isomerase: molecular genetic analysis of triplicate tryptophan pathway genes.

Phosphoribosylanthranilate isomerase (PAI) catalyzes the third step of the tryptophan biosynthetic pathway. Arabidopsis PAI cDNAs were cloned from a cDNA expression library by complementation of an Escherichia coli trpC- PAI deficiency mutation. Genomic DNA blot hybridization analysis detected three nonallelic genes encoding PAI in the Arabidopsis genome. DNA sequence analysis of cDNA and genomic clones indicated that the PAI1 and PAI2. All three PAI polypeptides possess an N-terminal putative plastid target sequence, suggesting that these enzymes all function in plastids. The PAI1 gene is flanked by nearly identical direct repeats of approximately 350 nucleotides. Our results indicate that, in contrast to most microorganisms, the Arabidopsis PAI protein is not fused with indole-3-glycerolphosphate synthase, which catalyzes the next step in the pathway. Yeast artificial chromosome hybridization studies indicated that the PAI2 gene is tightly linked to the anthranilate synthase alpha subunit 1 (ASA1) gene on chromosome 5. PAI1 was mapped to the top of chromosome 1 using recombinant inbred lines, and PAI3 is loosely linked to PAI1. cDNA restriction mapping and sequencing and RNA gel blot hybridization analysis indicated that all three genes are transcribed in wild-type plants. The expression of antisense PAI1 RNA significantly reduced the immunologically observable PAI protein and enzyme activity in transgenic plants. The plants expressing antisense RNA also showed two phenotypes consistent with a block early in the pathway: blue fluorescence under UV light and resistance to the anthranilate analog 6-methylanthranilate. The extreme nucleotide conservation between the unlinked PAI1 and PAI2 loci suggests that this gene family is actively evolving.     

TAIL-PCR的改良及其在分离小麦基因启动子中的应用

在经典TAIL-PCR(Thermal asymmetric interlaced PCR)的基础上, 对其进行了如下四处改进: 用10个碱基的RAPD引物代替16个碱基的随机兼并引物作为PCR中的随机引物; 将较低特异性循环的复性温度由44°C降至29°C; 增加5个高特异性反应循环, 减少5个较低特异性反应循环; 用单引物对第三轮PCR产物进行初步鉴定。利用改进的TAIL-PCR方法分离了小麦X基因的5′未知的侧翼序列, 与GUS基因融合后转入拟南芥, 通过组织化学检测分析表明分离到的5′侧翼序列具有启动子功能, 同时说明改进的TAIL-PCR能更好地应用到较复杂基因启动子的分离。     

rha1, a gene encoding a small GTP binding protein from Arabidopsis, is expressed primarily in developing guard cells.

The rha1 gene from Arabidopsis encodes a small GTP binding protein belonging to the Ypt/Rab family. Transgenic Arabidopsis plants containing the promoter region of the rha1 gene fused to the beta-glucuronidase (gus) reporter gene revealed gus expression limited mainly to the guard cells of stomata, the stipules, and the root tip of young plants. In flowering plants, expression was found predominantly in the receptacle and in guard cells of the different flower organs. High GUS activity could also be seen in callus tissue and developing seeds. No detectable activity was present in other plant tissues; activity could not be induced by various treatments. GUS activity was visualized histochemically using both 5-bromo-4-chloro-3-indolyl beta-D-glucuronide and a newly developed GUS substrate: Sudan II-beta-glucuronide. The latter precipitates as red crystals at the site of GUS activity. Results obtained by the gus analysis were confirmed by whole-mount mRNA in situ hybridization. A hypothesis for the function of the Rha1 protein is discussed.     

Light-dependent and tissue-specific expression of the H-protein of the glycine decarboxylase complex.

Glycine decarboxylase is a mitochondrial enzyme complex, which is the site of photorespiratory CO2 and NH3 release. Although the proteins that constitute the complex are located within the mitochondria, because of their intimate association with photosynthesis their expression is controlled by light. Comparisons of the kinetics of mRNA accumulation between the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase and the H-protein of glycine decarboxylase during the greening of etiolated Arabidopsis thaliana suggest that their expression is controlled in parallel. A genomic clone for the H-protein (gdcH) was isolated from Arabidopsis and sequenced. The upstream region from -856 to +62 was fused to the beta-glucuronidase (GUS) reporter gene, and this construct was transformed into tobacco. This 5' upstream regulatory region appears to control GUS expression in a manner very similar to that of the endogenous H-protein gene. Constructs with deletions in the 5' upstream region were transformed into tobacco. These deletions revealed that light-dependent and tissue-specific expression was largely controlled by a 259-bp region between -376 and -117 bp. This region contains several putative GT boxes with the GGTTAA consensus core sequence. Once these strong light-dependent elements were removed, a second level of control was revealed. In constructs in which the gdcH 5' regulatory region was shortened to -117 bp or less, there was more GUS activity in the roots than in the leaves, and in dark-grown plants than in light-grown plants. This suggests that more proximal control elements may be responsible for the constitutive low levels of gene expression noted in all nonphotosynthetic tissues.     

Analysis of rice Act1 5' region activity in transgenic rice plants.

The 5' region of the rice actin 1 gene (Act1) has been developed as an efficient regulator of foreign gene expression in transgenic rice plants. To determine the pattern and level of rice Act1 5' region activity, transgenic rice plants containing the Act1 5' region fused to a bacterial beta-glucuronidase (Gus) coding sequence were generated. Two independent clonal lines of transgenic rice plants were analyzed in detail. Quantitative analysis showed that tissue from these transgenic rice plants have a level of GUS protein that represents as much as 3% of total soluble protein. We were able to demonstrate that Act1-Gus gene expression is constitutive throughout the sporophytic and gametophytic tissues of these transgenic rice plants. Plants from one transgenic line were analyzed for the segregation of GUS activity in pollen by in situ histochemical staining, and the inheritance and stability of Act1-Gus expression were assayed in subsequently derived progeny plants.     

Structure and expression analyses of the S-adenosylmethionine synthetase gene family in Arabidopsis thaliana

The plant, Arabidopsis thaliana, contains two S-adenosylmethionine synthetase-encoding genes (sam). Here, we analyze the structure and expression of the sam-2 gene and compare it with the previously described sam-1 gene. Northern-blot analysis using gene-specific probes shows that both sam-1 and sam-2 are highly expressed in stem, root, and callus tissue. This similar expression pattern might be mediated by the presence of three highly conserved sequences in the 5' region of both sam genes. Using a chimeric beta-glucuronidase (GUS)-encoding gene, we show that in transgenic tobacco plants, 748 bp of 5' sam-1 sequences generate high GUS activity in the same type of tissues as previously observed in transgenic A. thaliana plants. A deletion analysis of these 5' sam-1 sequences indicates that 224 bp of 5' sam-1 sequences can still induce higher expression of the gene in stem and root relative to leaf. However, the level of expression is reduced when compared to the expression level obtained with the full-length promoter.     

Gene expression enhancement mediated by the 5′ UTR intron of the rice <Emphasis Type="Italic">rubi3</Emphasis> gene varied remarkably among tissues in transgenic rice plants

Introns are important sequence elements that modulate the expression of genes. Using the GUS reporter gene driven by the promoter of the rice (Oryza sativa L.) polyubiquitin rubi3 gene, we investigated the effects of the 5' UTR intron of the rubi3 gene and the 5' terminal 27 bp of the rubi3 coding sequence on gene expression in stably transformed rice plants. While the intron enhanced GUS gene expression, the 27-bp fused to the GUS coding sequence further augmented GUS expression level, with both varying among different tissues. The intron elevated GUS gene expression mainly at mRNA accumulation level, but also stimulated enhancement at translational level. The enhancement on mRNA accumulation, as determined by realtime quantitative RT-PCR, varied remarkably with tissue type. The augmentation by the intron at translational level also differed by tissue type, but to a lesser extent. On the other hand, the 27-bp fusion further boosted GUS protein yield without affecting mRNA accumulation level, indicating stimulation at translation level, which was also affected by tissue type. The research revealed substantial variation in the magnitudes of intron-mediated enhancement of gene expression (IME) among tissues in rice plants and the importance of using transgenic plants for IME studies.