The Influences of Two Plant Nuclear Matrix Attachment Regions (MARs) on Gene Expression in Transgenic Plants

Nuclear matrix attachment regions (MARs) are thought to influencegene expression by anchoring active chromatin to the proteinaceousnuclear matrix. In this study, two plant DNA fragments withstrong MAR activity were selected and tested for their effectson expression of a linked reporter gene in transgenic tobacco.One MAR was isolated from the 5' flanking region of a pea vicilingene previously reported to be expressed in a copy number-dependentmanner in transgenic tobacco. A second MAR was isolated fromthe genome of Arabidopsis thaliana by preselection for autonomouslyreplicating sequence (ARS) activity in yeast. Flanking copiesof the A. thaliana MAR stimulated median reporter gene expressionin transgenic plants by five to ten fold. Neither MAR significantlyreduced the variation in transgene expression between individualtransformants, or conferred copy number-dependence in gene expression. (Received July 24, 1997; Accepted November 10, 1997)     

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.     

A short synthetic MAR positively affects transgene expression in rice and Arabidopsis

Matrix Attachment Regions (MARs) are DNA elements that are thought to influence gene expression by anchoring active chromatin domains to the nuclear matrix. When flanking a construct in transgenic plants, MARs could be useful for enhancing transgene expression. Naturally occurring MARs have a number of sequence features and DNA elements in common, and using different subsets of these sequence elements, three independent synthetic MARs were created. Although short, these MARs were able to bind nuclear scaffold preparations with an affinity equal to or greater than naturally occurring plant MARs. One synthetic MAR was extensively tested for its effect on transgene expression, using different MAR orientations, plant promoters, transformation methods and plant species. This MAR was able to increase average transgene expression and produced integration patterns of lower complexity. These data show the potential of making well defined synthetic MARs and using them to improve transgene expression.     

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.     

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     

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.     

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。     

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.     

核基质结合区对转爬山虎芪合酶基因烟草中产生白藜芦醇的作用

采用核基质结合区(MARs)来提高转芪合酶基因(STS)烟草(Nicotianatabacum L.)中白藜芦醇产物的含量.MARs是细胞中能与核基质特异紧密结合的DNA片段,体外结合实验表明克隆自酵母的MARs序列能特异地与烟草核基质结合.芪合酶是白藜芦醇生物合成中的关键酶,用RT-PCR方法从川鄂爬山虎(Parthenocissus henryana(Hemsl.)Diels et Gilg)中克隆了与葡萄芪合酶基因有较高同源性的芪合酶编码区,将其置于CaMV35SΩ强启动子下,分别构建两侧带有MARs及不含MARs序列的表达载体,通过农杆菌介导转化烟草.Northern blot及HPLC等分析表明STS基因已整合至烟草染色体中并正常转录,且表达的外源芪合酶在烟草中可催化其底物合成白藜芦醇产物.与对照相比,MARs的存在使转芪合酶基因烟草中白藜芦醇的含量平均提高了约一倍.MARs在转芪合酶基因植物中的应用也为获得抗病性更强、白藜芦醇含量更高、更保健的转基因果蔬的研究奠定了基础.     

TM2, a novel strong matrix attachment region isolated from tobacco, increases transgene expression in transgenic rice calli and plants

Nuclear matrix attachment regions (MARs) are thought to influence the expression of the flanking genes. TM2, a new DNA fragment isolated from tobacco, can bind with the rice nuclear matrix in vitro. In this study, we investigated the effect of TM2 on transgene expression under the control of three different promoters in stably transformed rice calli and plants. The presence of TM2 flanking the transgene increased the expression of constructs based on the constitutive CaMV 35S and maize ubiquitin gene promoters in both resistant calli and transformed plants. The GUS expression directed by the photosynthetic-tissue-specific PNZIP promoter was also increased in photosynthetic tissues of transformants. However, TM2 did not change the gene expression pattern controlled by the PNZIP promoter. The effect of TM2 in transgenic plants was stronger than that in transgenic calli based on all three promoters. Our results indicate that TM2, as a novel strong MAR, can be used to increase the transgene expression levels in the whole plant or in particular tissues of monocotyledons.     

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.     

Suppression of transgene silencing by matrix attachment regions in maize: a dual role for the maize 5' ADH1 matrix attachment region

Matrix attachment regions (MARs) are DNA sequences that bind an internal nuclear network of nonhistone proteins called the nuclear matrix. Thus, they may define discrete gene-containing chromatin loops in vivo. We have studied the effects of flanking transgenes with MARs on transgene expression levels in maize callus and in transformed maize plants. Three MAR elements, two from maize (Adh1 5' MAR and Mha1 5' MAR) and one from yeast (ARS1), had very different effects on transgene expression that bore no relation to their affinity for the nuclear matrix in vitro. In callus, two of the MAR elements (Adh1 5' MAR and ARS1) reduced transgene silencing but had no effect on the variability of expression. In transgenic plants, Adh1 5' MAR had the effect of localizing beta-glucuronidase expression to lateral root initiation sites. A possible model accounting for the function of Adh1 5' MAR is discussed.     

The role of cell differentiation state and HMG-I/Y in the expression of transgenes flanked by matrix attachment regions

The tobacco nuclear matrix attachment region (MAR), RB7, has been shown to have a much greater effect on transgene expression in cultured cells than in transgenic plants. This is comparable to work in mouse systems showing that MARs have a positive effect on transgene expression in embryonic tissues but not adult tissues. There are several possible explanations for these observations. One is that cell differentiation state and proliferation rate can affect MAR function. We tested this possibility by initiating suspension cell cultures from well-characterized transgenic plants transformed with 35S::GUS with and without flanking MARs and then comparing GUS specific activity in the cell lines to those of the transgenic plants from which the cell lines were derived. If cell differentiation state and proliferation rate do affect MAR function, we would expect the ratio of transgene expression (cell suspensions : plants) to be greater in MAR lines than in control lines. This turned out not to be the case. Thus, it appears that MAR function is not enhanced simply because cells in culture divide rapidly and are not differentiated. Because in animal systems the chromosomal protein HMG-I/Y has been shown to be upregulated in proliferating cells and may have a role in MAR function, we have also examined the levels of the tobacco HMG-I/Y homolog by immunoblotting. The level of this protein does not differ between primary transformant cultured cells (NT-1) and Nicotiana tabacum plants (SR-1). However, a higher molecular weight cross-reacting polypeptide was found in nuclei from the NT-1 cell suspensions but was not detected in SR-1 leaf nuclei or cell suspensions derived from the SR-1 plants.     

Matrix attachment regions (MARs) enhance transformation frequency and transgene expression in poplar

We tested the value of a matrix attachment region (MAR) fragment derived from a tobacco gene for increasing the frequency of Agrobacterium-mediated transformation. A binary vector that carried a GUS reporter gene containing an intron and an nptII gene was modified to contain flanking MAR elements within the T-DNA borders. Vectors containing or lacking MARs were then used to transform tobacco, a readily transformabl e poplar clone (Populus tremula × P. alba), and a recalcitrant poplar clone (Populus trichocarpa × P. deltoides). MARs increased GUS gene expression approximately 10-fold in the two hybrid poplar clones and twofold in tobacco one month after cocultivation with Agrobacterium; MARs also increased the frequency of kanamycin-resistant poplar shoots recovered     

Approaching the Lower Limits of Transgene Variability

The inclusion of chicken lysozyme matrix-associated regions (MARs) in T-DNA has been demonstrated to reduce the variation in [beta]-glucuronidase (GUS) gene expression among first-generation transformed plants. The residual variation observed between transgenic plant lines with MARs at the T-DNA borders was investigated. By definition, any phenotypic variance between or within genetically identical plants is caused by random or environmental variation. This variation therefore sets a lower limit to the variation in GUS activities. The variance of GUS activity in offspring plant populations of genetically identical individuals was used as an estimate of environmental variation. For transgenic plants with MARs at the T-DNA borders, the variation between independent transformants could not be distinguished from the environmental variation. The variation could be attributed mainly to the variation in the GUS activity measurement. Therefore, the MAR element approached the maximal possible reduction of transgene variability given current technology and sample sizes. The role of MARs in offspring plants was evaluated by comparing such populations of transgenic plants for the magnitude of and variation in GUS activity. Pairwise comparisons showed that the presence of MARs reduced variation in offspring generations in the same manner as demonstrated for primary transformants. The populations carrying a doubled cauliflower mosaic virus 35S promoter-GUS gene tended to be more variable than the Lhca3.St.1 promoter-GUS gene-carrying populations. This tendency indicated an intrinsic susceptibility of the doubled cauliflower mosaic virus 35S promoter to variation. Homozygous plants were approximately twice as active as the corresponding hemizygous plants and tended to be more variable than the hemizygous plants. We hypothesized that the magnitude of environmental variations is related to a higher susceptibility to transgene silencing.     

The effect of MAR elements on variation in spatial and temporal regulation of transgene expression

The level of transgene expression often differs among independent transformants. This is generally ascribed to different integration sites of the transgene into the plant genome in each independently obtained transformant (position effect). It has been shown that in tobacco transformants expressing, for example, a cauliflower mosaic virus (CaMV) 35S promoter-driven -glucuronidase (GUS) reporter gene, these position-induced quantitative differences among individual transformants were reduced by the introduction of matrix-associated regions (MAR elements) on the T-DNA. We have previously shown by imaging of in planta firefly luciferase (luc) reporter gene activity that quantitative differences in transgene activity can be the result of either a variation in (1) level, (2) spatial distribution and/or (3) temporal regulation of transgene expression between independent transformants. It is not known which of these three different aspects of transgene expression is affected when the transgene is flanked by MAR elements. Here we have used the firefly luciferase reporter system to analyse the influence of MAR elements on the activity of a CaMV 35S-luc transgene in a population of independently transformed tobacco plants. Imaging of in planta LUC activity in these tobacco plant populations showed that the presence of MAR elements does not result in less variation in the average level of transgene expression between individual transformants. This result is different from that obtained previously with a 35S-GUS reporter gene flanked by MAR elements and reflects the differences in the stability of the LUC and GUS reporter proteins. Also the variation in spatial patterns of in vivo LUC activity is not reduced between independent transformants when the transgene is flanked by MAR elements. However, MAR elements do seem to affect the variation in temporal regulation of transgene expression between individual transformants. The potential effects of MAR elements on the variability of transgene expression and the relation to the stability of the (trans)gene product are discussed.     

The Influence of Matrix Attachment Regions on Transgene Expression in Transgenic Tobaccos

To investigate the effect of matrix attachment regions (MARs) on levels of transgene expression in transgenic plants, β-glucuronidase (GUS) gene ( uidA ) was flanked by the MARs isolated from the genome of pea ( Pisum sativum L.) to form plant expression vector. The plant expression vectors with and without MARs were transferred into tobaccos ( Nicotiana tabacum L.) via Agrobacterium -mediated transformation procedure. The results of GUS activity showed that MARs could increase levels of uidA gene expression, the mean GUS activity could be increased two fold compared with that of transformants without MARs, the highest GUS activity of transformant could arrive at five fold.     

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.