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Isolation of pea matrix attachment region and study on its function in transgenic tobaccos LI Xugang (李旭刚), ZHU Zhen (朱 祯), XU Junwang (徐军望), WU Qian (吴 茜) & XU Honglin (徐鸿林) Institute of Genetics, Chinese Academy of Sciences, Beijing 100101, China Correspondence should be addressed to Zhu Zhen (email: [email protected]) Received January 17, 2001

Abstract 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 has capability to increase expression level of gene in transgenic plants. Keywords: matrix attachment region, transgenic plants, β-glucuronidase, foreign gene expression.

Plant genetic engineering has created a novel approach to culture new kinds of fine cultivars. Many transgenic crops with economic merit have been gradually commercialized with the development of this subject. For an excellent transgenic plant, it is necessary to ensure the foreign target genes to be expressed efficiently in a predicable manner[1]. However, many factors, such as “position effect”, “DNA methylation”, “co-suppression” and “trans-inactivation” could lead to transgene silencing at transcriptional level, post-transcriptional level and other different stages of gene expression, which make the transgene expressed in an unpredictable manner. So it was important to adopt series of efficient strategies to increase transgene expression level and overcome gene silencing in the research of plant genetic engineering[2]. Some strategies, such as site-specific integrating of transgene, code alteration and other methods, have been used to overcome gene silencing in transgenic plants [1]. Recently, a good method by using matrix attachment regions (MARs) has been developed to prevent foreign gene silencing. MARs are AT-rich DNA fragments which can bind proteinceous matrix and make chromatin between MARs form DNA loop domain. Each chromatin loop is not only structural unit of chromosome but also unit of gene expression[3]. Transgenes flanked with MARs can be insulated to demarcate an independent regulatory domain by chromatin loop, thus transgene expres-

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sion is free from influence of “position effect”, and expression level and stability are increased in transgenic cells[3]. The prior experimental result has shown that variation of gene expression was decreased in primary transgenic plants and their progenies with MAR. MAR sequence could also decrease co-suppression effect on gene expression if transgene copies were no more than fifty[4]. The result published by Han et al. showed that tobacco MAR sequence could obviously increase GUS activity in transgenic poplar, and MAR had better effect in transgenic poplar than in transgenic tobacco[5]. In this research the DNA fragment containing characteristics sequence of MARs was isolated from pea genome[6]. To study whether the DNA sequence had biological function of MAR, the DNA sequence was cloned into plant expression vectors to be transferred into tobaccos. 1 1.1

Materials and methods Materials Escherichia coli DH5α, Agrobacterium tumefaciens LBA4404 and plasmids pBluescriptKS

(+) and pBin19 were preserved in our laboratory. Plasmids pSPGUSA, Ti plasmid pCNPT-II were constructed in our laboratory. Tobacco (Nicotiana tabacum L. cv NC89) was kept in our laboratory. Pea (Pisum sativum L. Grey) was bought from Institute of Vegetable, Chinese Academy of Agriculture. Restriction endonucleases and modification enzymes were bought from SABC, Biolab and Liuhetong Company. Tag enzyme (ExpandTM high fidelity PCR system) was bought from Boehringer Mannherim Company. Radioactive isotope α-32PdCTP was bought from Yahui Biotechnology Company. 1.2

Isolation of plant genome DNA The seven-day-old pea shoot genome DNA was isolated using the method of CTAB extraction procedure as described in ref. [7]. 1.3

PCR amplification of DNA sequence and cloning of the PCR product PCR amplification reaction was carried out according to the instruction of ExpandTM high fi-

delity PCR system. Reaction condition: denaturalization was at 94 ℃; annealing was at 60 ℃; extension was at 72 ℃, then heat preservation was at 72℃ for 10 min after cycles were finished. Two oligonucleotide primers were synthesized according to ref. [6], 5′ end primer P1: 5′ -CCATGCCTCACATGTTAATGTAC-3′ 3′ end primer P2: 5′ -CAACTCATCGGGAGATACTAGAG-3′ PCR products were recovered by low melting point agarose gel electrophoresis after being blunted with DNA polymerase I large fragment Klenow, then inserted into EcoR V sites of clone vector pBluescriptKS(+). Afterwards, the constructed vector was transformed into E. coli DH5 α with electric stimulus according to the protocol of electric stimulator of BIO-RAD Company. Then the white colony on the selective medium containing X-gal, IPTG and ampicillin was selected to obtain recombinant plasmid pBluePMAR.

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DNA sequencing analysis DNA fragment was sequenced according to the instruction of Sequenase Dye-Primer Sequencing Kit of Applied Biosystem Company in Shanghai Haojia Company of Biotechnology (fig. 1). 1.5

Construction of plant expression vectors To obtain plasmid pC2300PMAR, EcoR I/Cla I fragment containing MAR from pBluePMAR blunt ended with Klenow, was inserted into unique Pme I site immediate near RB of pCAMBIA2300, and then described orientation was selected. The plasmid pBluePMARNPT-II containing MAR-Pnos-nptII-Tnos was made from pBluePMAR and plasmid vector pBin19, and Pme I/Cla I fragment containing npt II gene (Pnos-npt-Tnos) from pBin19 was inserted into Hind III/Cla I site of plasmid pBluePMAR (Hind III ended was filled with Klenow). In order to make two MARs arranged in direct repeat, plasmid pBluePMARNPT-II was digested with EcoR I/Cla I to obtain DNA fragment containing MAR-nptII fragment (Cla I ended was blunted with Klenow), and inserted into Not I/EcoR I site of pBluescriptKS(+) (Not I ended was filled with Klenow), then plasmid pBluePMARNPT-II(-) was formed. The plasmid pCPMARNPT-II was made from pC2300PMAR and pBluePMARNPT-II(-), and Sal I/Sac I (only Sac I site was blunted with T4 DNA polymerase) fragment containing MAR-Pnos-nptII-Tnos from plasmid pBluePMARNPT-II(-), was inserted into BstX I/Xho I site of pC2300PMAR (BstX I ended was blunted with T4 DNA polymerase) to generate vector pCPMARNPT-II. The plant expression vectors pCNPT-IIGUSA, pCPMARNPT-IIGUSA were made from the intermediate plasmid pSPGUSA. The Pvu II/EcoR I fragment containing uidA gene under control of CaMV 35S promoter was inserted into Sma I/EcoR I site of pCPMARNPT-II, pCNPT-II respectively to generate plant expression vectors pCPMARNPT-IIGUSA (fig. 2(a)) and pCNPT-IIGUSA (fig. 2(b)). 1.6

Tobacco transformation and selection of regenerated plants Plant expression vectors pCNPT-IIGUSA, pCPMARNPT-IIGUSA and empty vector pCNPT-II were transferred into Agrobacterium tumefaciens LBA4404 by electroporation according to the instruction of E.coli Pulser apparatus (BIO-RAD). According to the method of ref. [5], the tobacco was transformed by the ameliorated leaf discs transformation method via Agrobacterium-mediate transformation procedure. Shoots were rooted on the medium containing 50 mg/L Kanamycin, then transferred to soil and grown in greenhouse. 1.7

Quantitative assay of GUS activity Quantitative GUS assay was carried out as described by Jefferson[8]. About 100 mg leaf was ground with 400 μL extraction buffer in Eppendorf tube and centrifuged. 10 μL of supernatant was added with 200 μL of substrate methylumbelliferylglucuronide (MUG), then incubated at 37°C for 30 min. GUS activity was measured at excitation wave of 360 nm and emission wave of 460 nm with a spectrofluorimeter (F-4010). Protein content was quantified using the standardized as-

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say method according to Bradford[9]. All GUS activities were expressed as picomole of methylumbelliferone (MU) per microgram of soluble protein per minute. 1.8

Southern blotting analysis Genome DNA was isolated from transgenic tobacco plants using the modified CTAB extrac-

tion procedure[7]. 10 μg of genome DNA was digested with EcoR I in appropriation reaction buffer at 37℃ overnight. DNA was then separated by electrophoresis in 0.8% agarose gel, and blotted onto Hybond N+ nylon membrane (Amersham) by capillary transfer. The membrane was pre-hybridized and hybridized according to Sambrook et al.[10]. The probe was 32P-labelled by random primer synthesis according to the manufacturer’s instructions. The hybridized membrane was washed with the solution containing 0.1 XSSC, 0.1% SDS solution, then exposed to X-ray film (Fuji). 2

Results

2.1

Cloning and analysis of MAR sequence The DNA fragment was amplified from template, pea shoot genome DNA. Primers P1 and P2 were designed according to the published sequence in Slatter’s article. Then isolated DNA sequence was cloned into plasmid pBluescriptKS(+) and sequenced. The result showed the amplified DNA fragment was 745 bp long (fig. 1), which contained several common consensus sequences, such as A-box or T-box (sequence in empty square), TATAA and AATATATTTT sequence (the sequence lined below) and topoisomerase II recognition sequence (sequences in black). But the amplified DNA fragment was shorter than anticipated DNA sequence[6], for the 115 bp-long repeat sequence was not found in DNA fragment that we isolated, and repeat sequence left was lined two. Two fragments located upstream and downstream of repeated sequence had 84% and 93% homology to the corresponding original sequence. 2.2

Construction of plant expression vector and tobacco transformation In order to determine whether the isolated sequence bore functions of MAR, a reporter uidA gene was used to analyze DNA function in tobaccos. In this research, the plant expression vector pCPMARNPT-IIGUSA was constructed with reporter uidA gene driven by CaMV35S promoter (fig. 2(a)). In plant expression vector two MARs were arranged in direct repeat because the effect of direct repeat was better than that of reverse repeat[11]. The control vector pCNPT-IIGUSA lacking MAR only contained chimertic uidA gene construction (fig. 2(b)). The empty vector pCNPT-II having no uidA gene was used as negative control (fig. 2(c)). 2.3

Analysis of GUS activity of transgenic plants Quantitative GUS activity of tobacco leaves was measured according to Jefferson. In this experiment, the third expansion leaf was measured to decrease experimental error. GUS activity of 42 transgenic plants with pCPMARNPT-IIGUSA and pCNPT-IIGUSA was measured, and that of 10 transgenic plants from empty vector pCNPT-II and 10 untransformated tobaccos was also

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Analysis of MAR sequence. The sequence in empty square is A-box, T-box; the sequence lined below is AT

sequence; the shadow sequence is topoisomerase II; the black sequences are primers; the left sequence is lined two.

Fig. 2. Diagram of plant expression vectors. (a) pCPMARNPT-IIGUSA; (b) pCNPT-IIGUSA; (c) pCNPT-II. P-35S, Promoter of cauliflower mosaic virus (CaMV)35S protein; nptII, plant selective marker gene nptII; uidA, reporter gene uidA; T-nos, terminator of nopaline synthase (Nos) gene; LB, RB, left and right borders of T-DNA.

measured. As a result, the overall GUS expression level was higher in transgenic population of plasmid pCPMARNPT-IIGUSA than that of pCNPT-IIGUSA transgenic population (fig. 3(a),(b)). The average GUS activity with MAR was two times higher than that of tobaccos without MAR

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(fig. 3(c)), and the highest GUS activity was 12.77 nmol MU mg−1 protein min−1. In transgenic population with empty vector, the basal GUS activity was the same as that of untransformated tobaccos (fig. 3(c)).

Fig. 3. GUS activity of transgenic tobaccos. GUS activity of transgenic plant leaves was measured at the excitation wave of 360 nm and emission wave of 460 nm. Each column represents individual GUS activity of transgenic tobacco. (a) GUS activity of tobaccos with pCPMARNPT-IIGUSA; (b) GUS activity of tobaccos with pCNPT-IIGUSA; (c) average GUS activity of transgenic tobaccos, pCPMARNPT-IIGUSA, pCNPT-IIGUSA, pCNPT-II, transgenic tobaccos with corresponding vectors. CK, untransformated tobaccos.

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DNA blot analysis of transgenic plants Tobacco genome DNA was separated by electrophoresis after being digested with enzyme EcoR I, then was transferred onto Hybond N+ nylon membrane. A 2.0 kb BamH I/Sac I DNA fragment containing coding sequence of uidA gene from plasmid pSPGUSA was used as probe, which was labelled with α-32PdCTP and hybridized with membrane. As the results shown in fig. 4, in 17 transgenic plants analyzed with Southern blotting, the numbers of transgenic plants with plasmid pCPMARNPT-IIGUSA and pCNPT-IIGUSA were 14 and 3 respectively. In 17 transgenic plants, DNA hybridization bands all emerged. The result showed that uidA gene had already integrated into genome of tobaccos.

Fig. 4. Southern blotting analysis of transgenic tobaccos. 1—3, Southern blotting of tobaccos with pCNPT-IIGUSA, whose genome DNA was digested with EcoR I; 5—18, Southern blotting of tobaccos with pCPMARNPT-IIGUSA, whose genome DNA was digested with EcoR I; ck, untransformated plant genome DNA digested with EcoR I, gus, positive uidA gene. The band signed by arrow is 2.0 kb.

3

Discussion

It has been an efficient strategy to overcome transgene silencing by adopting matrix attachment region, which can increase expression levels and expression stability of transgene. Matrix attachment regions are specific DNA fragments in eukaryotic chromatin, usual in size from several hundreds bp to several kilob. The primary character of MARs sequences is AT rich DNA sequence, and contains several consensus sequences, such as A-box (AATAAAAA/CAA), T-box (TTTTATTTTT), TATAAA motif and topoisomerase II recognition sequence. Because of specific DNA structure formed by these DNA motifs, MARs can interact with cell matrix and make chromatin between MARs to form DNA loop domain[12]. Each chromatin loop is not only structural unit of chromosome but also unit of gene expression. Genes in chromatin loop flanked with MARs are free from effect of host chromatin. Based on this trait of MARs, transgenes can be insulated by chromatin loop if they are

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flanked with MARs to demarcate a regulatory domain and gene being free from influence of “position effect” and gene silencing induced by other factor, transgene silencing in transgenic plants, thus increase transgene expression level and stability in transgenic cells[3]. The MARs isolated from different organisms can increase gene expression in primary transformants and progenies of transgenic plants[11, 13,14]. In this research, a 860 bp-long MAR sequence was going to be amplified with designed primers according to the MAR sequence located downstream of pea plastocyanin gene isolated by Slatter et al. The sequencing result showed that the 115 bp-long repeat sequence was deleted in isolated DNA sequence compared with anticipated DNA fragment, but amplified DNA fragment was still AT rich sequence, and also contained characteristic motifs of MAR, such as A-box (AATAAAAA/CAA), T-box (TTTTATTTTT), TATAAA DNA motif and topoisomerase II recognition sequence (fig. 1). According to Slatter et al., they only isolated the MAR from pea genome, but the function of MAR was not determined. In order to make sense the ability of MAR-like sequence, we constructed plant expression vector pCPMARNPT-IIGUSA whose reporter gene was flanked with the MAR-like sequence (fig 2(a)). Tobaccos were selected and GUS activity of transgenic tobaccos obtained via Agrobacterium-mediated transformation method was measured. GUS activity of total 42 different transgenic tobaccos with plant expression vectors pCPMARNPTIIGUSA and pCNPT-IIGUSA respectively was also measured. The overall GUS activity was higher in transgenic plants with MAR than in transgenic plants without pea MAR sequence (fig. 3(a), (b)), and average GUS activity level was two times higher in the presence of MAR (fig. 3(c)). As for overcoming gene silencing, the number of transgenic plants indicating transgene silencing in plants with expression vector pCPMARNPT-IIGUSA was much less than in plants with expression vector pCNPT-IIGUSA. The result proved that MAR could not only increase gene expression level but also overcome transgene silencing. And variation of gene expression was decreased twofold in plant with expression vector pCPMARNPT-IIGUSA compared with that of plant expression pCNPT-IIGUSA. However, the gene expression level had been influenced not only by position effect, but also by DNA methylation and repeat sequence. So in some degree MAR could overcome gene silencing. This was the reason why GUS activity of some pCPMARNPT- IIGUSA was lower than that of pCNPT-IIGUSA transgenic tobaccos. The molecular analysis, Southern blotting indicated that uidA gene copies integrating into transgenic tobacco plants were not uniform (fig. 4). But it was statistically obvious that MAR could increase gene expression level. Now, genetic analysis of progeny of silencing plants is being done in order to understand deeply whether the effect of MAR on gene silencing depended on gene copies. From the results cited above, the MAR contained not only characteristic sequence of MAR but also biological function of MAR. So it was proved that isolated MAR has activity to increase gene expression in transgenic plants. It has been claimed that the interaction between MAR and matrix is conservative in evolution. MARs and matrix isolated from different species can interact each other in plants, MAR of soy-

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bean heat shock gene can bind matrix of tobacco. MARs and matrix isolated from animals and plants can also interact. These results sufficiently showed the conservation between interaction of MAR and matrix[11]. Our result suggested that pea MAR could increase foreign expression level in tobaccos, that pea MAR could interact with tobacco matrix, implying that pea MAR could have capabilities in other plants. It will be important to develop transgenic plants integrated with high and stable expression of transgenes. Acknowledgements This work was supported by the National High Science and Technology Program, National Natural Science Foundation of China (Grant Nos. 39989001 & 39880023), and National Special Program for Research and Industrialization of Transgenic Plants.

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