Isolation of pea matrix attachment region and study on its function in transgenic tobaccos

A DNA fragment containing consensus sequence of matrix attachment region (MAR) has been isolated from pea genome. Compared with original DNA sequence, one 115 bp-long repeat sequence is deleted in the obtained DNA sequence. DNA fragments located upstream and downstream of repeat DNA sequence respectively share 84% and 93% homology to the corresponding original sequence, and contain A-box or T-box and TATAA sequence, which is characteristics short sequence of MARs. To test the function of the DNA sequence, the plant expression vectors in which β-glucuronidase gene (GUS, uidA) was used as reporter gene were constructed and transferred into tobaccosvia Agrobacterium- mediated transformation procedure. Quantitative GUS assay showed that the average level of uidA expression was increased twofold for the presence of MAR, and the highest level of GUS activity of transgenic plants could be increased six times. The results cited above suggest that the isolated DNA sequence contains consensus sequence of MARs and has capability to increase expression level of gene in transgenic plants.     

Elevation of Transgene Expression Level by Flanking Matrix Attachment Regions (MAR) is Promoter Dependent: A Study of the Interactions of Six Promoters with the RB7 3′ MAR

We have analyzed effects of a matrix attachment region (MAR) from the tobacco RB7 gene on transgene expression from six different promoters in stably transformed tobacco cell cultures. The presence of MARs flanking the transgene increased expression of constructs based on the constitutive CaMV 35S, NOS, and OCS promoters. Expression from an induced heat shock promoter was also increased and MARs did not cause expression in the absence of heat shock. There was also no effect of MARs on the pea ferredoxin promoter, which is not normally expressed in this cell line. Importantly, most transgenes flanked by RB7 MAR elements showed a large reduction in the number of low expressing GUS transformants relative to control constructs without MARs.     

Isolation of pea matrix attachment region and study on its function in transgenic tobaccos

A DNA fragment containing consensus sequence of matrix attachment region (MAR) has been isolated from pea genome. Compared with original DNA sequence, one 115 bp-long repeat sequence is deleted in the obtained DNA sequence. DNA fragments located upstream and downstream of repeat DNA sequence respectively share 84% and 93% homology to the corresponding original sequence, and contain A-box or T-box and TATAA sequence, which is characteristics short sequence of MARs. To test the function of the DNA sequence, the plant expression vectors in which β-glucuronidase gene (GUS, uidA) was used as reporter gene were constructed and transferred into tobaccos via Agrobacterium-mediated transformation procedure. Quantitative GUS assay showed that the average level of uidA expression was increased twofold for the presence of MAR, and the highest level of GUS activity of transgenic plants could be increased six times. The results cited above suggest that the isolated DNA sequence contains consensus sequence of MARs and     

烟草MARs的分离及其功能分析

从烟草基因组中克隆到两条新的MAR片段（M14和M17）,序列分析表明,它们具有90%AT-box,A-box,T-box,碱基非配对区域,拓扑异构酶Ⅱ识别位点,弯曲DNA序列,复制起始序列和ATATTT等典型的MAR序列特征,并与原有MAR序列的特征不同。将它们分别构建到植物表达载体pCAMBIA2301 GUS基因（uidA）表达盒一侧及两侧,通过农杆菌介导转化烟草。组织化学染色法定性检测GUS活性表明,带有M14和M17的uidA基因在转基因烟草中稳定表达。GUS活性的定量检测表明,表达载体上uidA基因一端或两端连接有MAR的转化烟草中,GUS的表达水平与对照相比都有了明显提高,而uidA基因两侧连有MAR的载体提高表达水平的效果优于一端连有MAR的载体,可使GUS活性增强3.14倍,但不同转化个体之间表达水平的差异仍然明显。上述结果表明,所得DNA序列为两条新的MAR片段,并且具有提高转基因表达水平的功能。     

Effect of nuclear matrix attachment regions on transgene expression in tobacco plants

Matrix attachment regions (MARs) are thought to participate in the organization and segregation of independent chromosomal loop domains. Although there are several reports on the action of natural MARs in the context of heterologous genes in transgenic plants, in our study we tested a synthetic MAR (sMAR) with the special property of unpairing when under superhelical strain, for its effect on reporter gene expression in tobacco plants. The synthetic MAR was a multimer of a short sequence from the MAR 3' end of the immunoglobulin heavy chain (IgH) enhancer. This sMAR sequence was used to flank the beta-glucuronidase (GUS) reporter gene within the T-DNA of the binary vector pBI121. Vectors with or without the sMARs were then used to transform tobacco plants by Agrobacterium tumefaciens. Transgenic plants containing the sMAR sequences flanking the GUS gene exhibited higher levels of transgene expression compared with transgenic plants which lacked the sMARs. This effect was observed independently of the position of the sMAR at the 5' side of the reporter gene. However, variation of the detected transgene expression was significant in all transformed plant populations, irrespective of the construct used.     

Meiotic stability of transgene expression is unaffected by flanking matrix-associated regions

DNA repeats are associated with gene instability and silencing phenomena in plants. Therefore, the presence of a direct repeat of matrix-associated region (MAR) DNA, that considerably reduced position effects between independent transformants, may increase (epi)genetic instability. To investigate the influence of such a repeat on the stability of the expression of embedded transgenes, the meiotic stability of transgene expression was assessed in eighteen homozygous 1-locus transgenic tobacco lines carrying the kanamycin resistance (NPTII) and the -glucuronidase (GUS) gene. Half of the lines carry a 3 kb direct repeat of MAR DNA flanking the transgenes. Large progeny populations, totalling over a million seedlings, were screened for kanamycin resistance with the help of a newly developed high-density seedling screen. Kanamycin-sensitive seedlings were detected in selfed progeny at a frequency of 0.5-5.9×10^-4. The frequency became as high as 2×10^-2 when embryo development occurred under heat and/or drought stress. In backcrossed progeny only, a joint loss of NPTII and GUS gene expression was observed at an average frequency of 2.9×10^-5. In selfed and backcrossed progeny we observed similar frequencies of reversion to kanamycin sensitivity, indicating that epigenetic silencing mechanisms rather than MAR repeat-related homologous recombination underlie the reversal to kanamycin sensitivity. Different lines, hence different areas of the tobacco genome, differed in their genetic stability. No significant differences in reversal frequencies were apparent between lines with or without the MAR elements. The use of the MAR repeat is, therefore, not compromised by any increased (epi)genetic instability.     

Functional analysis of BnMAR element in transgenic tobacco plants

Scaffold/matrix attachment regions (S/MARs) are defined as genomic DNA sequences, located at the physical boundaries of chromatin loops. Previous reports suggest that S/MARs elements may increase and stabilize the expression of transgene. In this study, DNA sequence with MAR characteristics has been isolated from B. napus . The BnMARs sequence was used to flank the CaMV35S-GUS-NOS expression cassette within the T-DNA of the plant expression vector pPZP212. These constructs were introduced into tobacco plants, respectively and the GUS reporter gene expression was investigated in stably transformed plants. When the forward BnMARs sequence was inserted into the upstream of CaMV35S promoter, the average GUS activities were much higher than those without BnMARs in transgenic tobacco. The GUS expression of M(+)35S:GUS, M(+)35S:GUSM(+) and M(+)35S:GUSM(−) constructs increased average 1.0-fold, with or without BnMARs located downstream of NOS. The GUS expression would not be affected when reverse BnMARs sequence inserted whether upstream of CaMV35S promoter or downstream of NOS. The GUS expression was affected a little when reverse BnMARs sequence was inserted the downstream of NOS and BnMARs could not act by serving as of promoter. The results showed that the presence of forward BnMARs sequence does have an obvious impact on enhancing downstream gene expression and its effect is unidirectional.     

High transformation frequency of tobacco and rice via Agrobacterium-mediated gene transfer by flanking a tobacco matrix attachment region

Nuclear matrix attachment regions (MARs) are suggested to regulate chromatin structure and influence the expression of flanking genes. Our previous study showed that TM2, a new DNA fragment isolated from tobacco, can bind with the rice nuclear matrix in vitro and increase transgene expression in vivo. Here, we investigated the role of TM2 MAR in improving transformation frequency of Agrobacterium -mediated transformation in tobacco and rice. The gusA reporter gene flanked by TM2 MAR in pBI121 and pCAMBIA-1301 vectors was controlled by a constitutive promoter or a photosynthetic tissue-specific promoter. The presence of TM2 MAR in different expression cassettes significantly increased the numbers of kanamycin-resistant tobacco shoots, hygromycin-resistant rice calli and shoots. Seeds from the independent transgenic lines with TM2 MAR can germinate normally on the medium containing 500 mg l⁻¹ kanamycin, whereas none of the seeds from the transgenic lines without TM2 MAR survived. Furthermore, RNA gel blot analysis revealed that nptII messenger RNA levels are more abundant in the independent transgenic lines with TM2 MAR than in the lines without TM2 MAR. Altogether, these data reveal a possible mechanism that TM2 MAR improves the transformation frequency by increasing nptII gene expression.     

A tobacco matrix attachment region reduces the loss of transgene expression in the progeny of transgenic tobacco plants

The RB7 matrix attachment region (MAR), when flanking a uidA (GUS) reporter gene, has been previously shown to increase uidA gene expression by 60-fold in stably transformed tobacco suspension cell lines. We have now used the same co-transformation procedure to determine the effect of flanking MARs on uidA gene expression in tobacco plants. The neomycin phosphotransferase selection gene and uidA reporter gene on separate plasmids were co-transformed into seedlings by microprojectile bombardment. In primary transgenic plants, the average uidA expression in plants with MARs was twofold greater than in control plants without MARs, but there was no effect on variation of expression. GUS activity was not proportional to the number of integrated uidA transgenes over the entire range of copy numbers. However, in the lower part of the copy number range, MAR lines show a tendency for expression to increase with copy number. Transgene expression in backcross progenies of the MAR-containing lines averaged threefold higher than in control progenies. MARs also reduced the loss of transgene expression in the BC₁ generation. Sixty-three per cent of the 21 MAR-containing primary transformants, but only 20% of the 14 control primary transformants, produced backcross progenies in which no loss of transgene expression was observed. These observations are discussed in the context of homology-dependent gene silencing.     

The Populus PTD promoter imparts floral-predominant expression and enables high levels of floral-organ ablation in Populus,Nicotiana and Arabidopsis

We evaluated the utility of the promoter from the Populus PTD gene—homologous to the MADS box genes DEFICIENS and APETALA3—to genetically engineer reproductive sterility. Floral-predominant expression was confirmed via GUS reporter assays in two heterologous species (Arabidopsis and tobacco) and in an early-flowering poplar genotype. Using the PTD promoter to direct expression of the disarmed cytotoxin DTA resulted in sterile plants with otherwise normal growth at high frequency in all three species. Biomass production in greenhouse-grown, morphologically normal tobacco cytotoxin lines was indistinguishable from lines lacking the cytotoxin gene, confirming strong floral specificity of the promoter. These results suggest that the poplar PTD promoter may prove useful for transgene confinement without detrimental effects on yield.     

Functional Analysis of Two Matrix Attachment Region (MAR) Elements in Transgenic Maize Plants

Matrix attachment regions (MARs) are binding sites for nuclear scaffold proteins in vitro, and are proposed to mediate the attachment of chromatin to the nuclear scaffold in vivo. Previous reports suggest that MAR elements may stabilize transgene expression. Here, we tested the effects of two maize MAR elements (P-MAR from the P1-rr gene, and Adh1-MAR from the adh1 gene) on the expression of a gusA reporter gene driven by three different promoters: the maize p1 gene promoter, a wheat peroxidase (WP) gene promoter, or a synthetic promoter (Rsyn7). The inclusion of P-MAR or Adh1-MAR on P::GUS transgene constructs did not reduce variation in the levels of GUS activity among independent transformation events, nor among the progeny derived from each event. The Adh1-MAR element did not affect GUS expression driven by the WP promoter, but did modify the spatial pattern of expression of the Rsyn7::GUS transgene. These results indicate that, in transgenic maize plants, the effects of MAR elements can vary significantly depending upon the promoter used to drive the transgene.     

Matrix attachment region elements have small and variable effects on transgene expression and stability in field‐grown Populus

Matrix attachment regions (MARs) are thought to buffer transgenes from the influence of surrounding chromosomal sequences, and therefore to reduce transgene silencing and variation in expression. The statistical properties of more than 400 independent transgenic events produced in Populus, with and without flanking MAR elements from the tobacco root gene RB7, were analysed. The expression of two reporter genes in two poplar clones during three phases of vegetative growth, and the association of T‐DNA characteristics with expression, was examined. It was found that MARs did not show a consistent effect on transgene expression levels; they had no effect on the green fluorescent protein (GFP) reporter gene, but reduced expression in the Basta resistance (BAR) reporter gene by 23%. The presence of MARs reduced expression variability within transformant populations, apparently by reducing the number of silenced or weakly expressing events. Transgene expression was highly stable over vegetative growth cycles that spanned 3 years of growth in the glasshouse and field, but MARs showed no association with the strength of correlations in expression over the years. Nonetheless, MARs increased the correlation in expression between a p35S::GFP and prbcS::BAR transgene linked on the same vector, but the effect was small and varied between the years. The presence of MARs had no effect on the transgene copy number, but was positively associated with T‐DNA truncations, as well as with the formation of direct over inverted repeats at the same chromosomal locus.     

Anaerobic induction of the maize<Emphasis Type="Italic"> GapC4</Emphasis> promoter in poplar leaves requires light and high CO<Subscript>2</Subscript>

The maize (Zea mays L.) glyceraldehyde-3-phosphate dehydrogenase gene 4 (GapC4) promoter confers anaerobic gene expression in tobacco (Nicotiana tabacum L.), potato (Solanum tuberosum L.) and Arabidopsis thaliana (L.) Heynh. Here we have investigated its expression in hybrid poplar (Populus tremula × P. alba). Our results show that the promoter is not expressed in leaves and stems under normoxic conditions while anaerobiosis induces reporter gene expression in leaves up to a level observed for the STLS-1 promoter from potato that is shown to confer leaf-specific gene expression in transgenic poplar. Anaerobic induction is cell autonomous and requires a CO₂ atmosphere and light. As in tobacco, the GapC4 promoter in poplar is wound inducible. The induction by CO₂ and light may reflect a natural situation because flooding, a natural cause of anaerobiosis, is often accompanied by high CO₂ concentrations in the floodwater. Our results show that the GapC4 promoter is suitable as an anaerobic reporter and as an inducible gene expression system in poplar.Abbreviations CaMV cauliflower mosaic virus - GapC4 glyceraldehyde-3-phosphate dehydrogenase gene 4 - GUS -glucuronidase - 4-MU methylumbelliferone - STLS-1 stem- and leaf-specific promoter 1     

Matrix attachment regions increase transgene expression levels and stability in transgenic rice plants and their progeny

To investigate the effect of matrix attachment regions (MARs) on transgene expression levels and stability in cereal crops, we generated 83 independent transgenic rice callus lines containing a gusA expression cassette either as a simple expression unit, or flanked with MARs from tobacco (Rb7) or yeast (ARS1). Transgenic rice plants were regenerated from these callus lines and analysed at the structural and expression levels over two generations. In the first generation (T₀), both Rb7 and ARS1 MARs significantly increased transgene expression levels. In the populations of plants containing MARs, we observed a significant reduction in the number of non-expressing lines compared to the population of plants without MARs. However, variation in β-glucuronidase (GUS) expression levels between independent lines was similar both in the presence and absence of flanking MARs. In the presence of MARs, GUS activity increased in proportion to transgene copy number up to 20 copies, but was generally reduced in lines carrying a higher copy number. In the population of plants without MARs, there was no correlation between expression level and transgene copy number. In the second generation (T₁), transgene expression levels were significantly correlated with those of the T₀ parents. The Rb7 MARs significantly improved the stability of transgene expression levels over two generations, and therefore appear to offer protection against transgene silencing. Our study shows that the exploitation of MARs may be an important strategy for stabilising transgene expression levels in genetically engineered cereals.     

Enhancement of secondary xylem cell proliferation by Arabidopsis cyclin D overexpression in tobacco plants

Secondary xylem is composed of daughter cells produced by the vascular cambium in the stem. Cell proliferation of the secondary xylem is the result of long-range cell division in the vascular cambium. Most xylem cells have a thickened secondary cell wall, representing a large amount of biomass storage. Therefore, regulation of cell division in the vascular cambium and differentiation into secondary xylem is important for biomass production. Cell division is regulated by cell cycle regulators. In this study, we confirm that cell cycle regulators influence cell division in the vascular cambium in tobacco. We produced transgenic tobacco that expresses Arabidopsis thaliana cyclin D2;1 (AtcycD2;1) and AtE2Fa-DPa under the control of the CaMV35S promoter. Each gene is a positive regulator of the cell cycle, and is known to influence the transition from G1 phase to S phase. AtcycD2;1-overexpressing tobacco had more secondary xylem cells when compared with control plants. In order to evaluate cell division activity in the vascular cambium, we prepared a Populus trichocarpa cycB1;1 (PtcycB1;1) promoter containing a destruction box motif for ubiquitination and a β-glucuronidase-encoding gene (PtcycB1;1pro:GUS). In transgenic tobacco containing PtcycB1;1pro:GUS, GUS staining was specifically observed in meristem tissues, such as the root apical meristem and vascular cambium. In addition, mitosis-monitoring plants containing AtcycD2;1 had stronger GUS staining in the cambium when compared with control plants. Our results indicated that overexpression of AtcycD enhances cell division in the vascular cambium and increases secondary xylem differentiation in tobacco. Key message We succeeded in inducing cell proliferation of cambium and enlargement of secondary xylem region by AtcycD overexpression. We also evaluated mitotic activity in cambium using cyclin-GUS fusion protein from poplar.     

核基质结合区的位置对转基因表达的影响

为研究核基质结合区 (MAR）序列不同插入位置对转基因表达作用的影响,PCR扩增人β 珠蛋白MAR分别插入到含氯霉素乙酰转移酶（chloramphenicol acetyltransferase,CAT）报告基因真核表达载体pCATG表达盒两侧、5′端及3′端.酶切鉴定后,用阳离子聚合物转染CHO细胞,G418筛选出阳性细胞克隆,ELISA分析CAT基因的表达水平,半定量PCR分析CAT基因相对拷贝数.结果表明,表达盒两侧含MAR序列的载体能提高介导的转基因表达水平平均提高10.4倍,5′端含MAR序列的载体表达水平平均提高3.9倍,3′端含MAR序列的载体反而降低转基因表达水平.5′端含MAR序列的表达载体其转基因相对拷贝数高于其它两组载体的基因拷贝数,转基因表达量与基因拷贝数不成正比.