Plant Biotechnol Rep DOI 10.1007/s11816-009-0089-4
ORIGINAL ARTICLE
T1 transgenic tobacco plants carrying multicopy T-DNAs at the same locus exhibit various expression levels of transgenes Sung Ran Min Æ Seyed Javad Davarpanah Æ Youn-il Park Æ Jae Heung Jeon Æ Jae Sun Moon Æ Jang Ryol Liu Æ Won Joong Jeong
Received: 16 January 2009 / Accepted: 26 March 2009 Ó Korean Society for Plant Biotechnology and Springer 2009
Abstract Multicopy integration of transgenes usually causes gene silencing of the transgenes. To investigate gene silencing of multicopy transgenes, we generated tobacco transgenic plants with T-DNA carrying the HygR and tflA genes. Southern blot analyses revealed that the transgenic plant lines 26 and 28 had a single and three copies of T-DNA, respectively. Northern blot analyses indicated that both HygR and tflA genes were expressed at a high level in T0 and T1 generations of line 26. However, the HygR and tflA genes were expressed at a high and low level, respectively, in the T0 generation of line 28. Furthermore, the HygR and tflA genes were expressed either at a high or low level in the T1 progeny of line 28, depending on individual plants. Restriction digestion analyses indicated that three copies of T-DNA in line 28 were arranged in head-to-head and tail-to-tail repeats at the same locus in the genome. Overall results suggest that multicopy transgenes cause silencing of the transgenes in a more complicated manner than transgenes are simply silenced in all progeny of the T1 generation. Keywords Nicotiana tabacum T-DNA repeat Transgene arrangement Transgene expression Silencing
S. R. Min S. J. Davarpanah J. H. Jeon J. S. Moon J. R. Liu W. J. Jeong (&) Plant Systems Engineering Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), 111 Gwahangno, Yuseong-gu, Daejeon 305-806, South Korea e-mail:
[email protected] S. J. Davarpanah Y.-i. Park Department of Biology, Chungnam National University, Daejeon 305-764, South Korea
Introduction It is desirable that the expression of transgenes can be predictable and stable in transgenic plants. However, expression levels of transgenes vary among transgenic lines even when the same vector is used for transformation (Birch 1997; Butaye et al. 2004; Kamoi et al. 2008). Low or no transgene expression, which is known as transgene silencing, is observed in the next or later generations in a large portion of transgenic plants that were selected to have a high expression of transgene at initial stages (Finnegan et al. 2001; Hannon 2002; Iwai et al. 2002). It was reported that 80% of transgenic lines exhibited abnormally low expression of the GUS gene in Arabidopsis and that 56–80% of T2 or T3 generation showed no expression of transgenes in Arabidopsis and wheat (Anand et al. 2003; Meza et al. 2001), indicating that only a small portion of transgenic plants stably express the foreign gene over generations. A transgene integrated into the plant genome in the form of multicopy is silenced more frequently than when in the form of a single copy (Hobbs et al. 1993; Jorgensen et al. 1996; Wang and Waterhouse 2000). Furthermore, it has been reported that transgene is silenced when multicopies of T-DNA are integrated in tandem repeat or inverted repeat arrangements at the same locus of the plant genome (Chan et al. 2006; De Buck et al. 1999; Martienssen 2003; Muskens et al. 2000). It is postulated that abnormal transcripts from repeat arrangements trigger to induce gene silencing machinery to keep degradation of the abnormal RNAs (PTGS) and then sometimes to block the transcription of the locus (TGS) (Brodersen and Voinnet 2006; Fojtova´ et al. 2003, 2006; Hannon 2002; Mette et al. 1999; Muskens et al. 2000; Sijen et al. 2001).
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In this study, we demonstrated that transgenic tobacco plants carrying multicopy transgenes at the same locus exhibited different expression levels of transgenes in each progeny of the T1 generation. These results suggest that multicopy transgenes cause silencing of the transgenes in a more complicated manner than transgenes are simply silenced in all progeny of the T1 generation.
Materials and methods Production of transgenic plants Transgenic tobacco plants were produced by co-cultivation of Agrobacterium tumefaciens LBA 4404 carrying pCamLA::tflA, which has two genes including HygR (hygromycin phosphotransferase) as a selection marker for hygromycin and tflA (Toxoflavin lyase) gene on T-DNA (Fig. 1a). Hygromycin-resistant plants were generated on MS medium containing 50 mg l-1 hygromycin and then subjected to PCR to confirm the presence of the tflA gene. Transgenic plants were transferred to potting soil and then grown to maturity in a growth chamber. T1 seeds were obtained by self-pollination and then placed on medium containing 50 mg l-1 hygromycin. Germinated plants were also transferred to potting soil for molecular analysis.
DNA and RNA analyses Total RNA was prepared using Tri-Reagent (Invitrogen), following the manufacturer’s instructions. Approximately 20 lg total RNA was electrophoresed on 1% agarose gel containing 5.1% (v/v) formaldehyde and then blotted onto nylon membrane (Zeta-Probe GT genomic tested blotting membranes; Bio-Rad) in 20X SSC. Genomic DNA was isolated from nontransformed and transformed tobacco leaf tissues by the method of Dellaporta et al. (1983). Each 50 lg genomic DNA digested with EcoRI, HindIII, KpnI, XbaI, and XhoI were separated on 0.8% (w/v) agarose gels and blotted onto nylon membranes in 109 SSC. The 538 bp tflA DNA fragment and 1 kb HygR DNA fragment amplified by PCR were used as a probe and then labeled with [32P]dCTP using the Random Primed DNA Labeling kit (Boehringer Mannheim). Prehybridization and hybridization were carried out in 0.25 M sodium phosphate (pH 7.2) and 7% (w/v) SDS solution at 65°C for overnight. The membranes were washed in 20 mM sodium phosphate (pH 7.2) and 5% (w/v) SDS at 65°C for 15 min, then washed in 20 mM sodium phosphate (pH 7.2) and 1% (w/v) SDS at 65°C for 15 min. The membranes were exposed using Imaging Plate (Fujifilm, Japan) at room temperature or X-ray film at -70°C and then the film was developed.
Results Unstable expression of transgene in transgenic lines Regenerated hygromycin resistant plants were confirmed to carry tflA gene by PCR (data not shown). In each of T0 transgenic lines, Northern blot analysis revealed various expression levels of the transgenes and Southern blot analysis detected one to several copies of T-DNA integrated into the genome (Fig. 1b, c). Transgene expression in T1 generation
Fig. 1 Various expressions of transgenes in transgenic lines of N. tabacum. Transgenes insertion and expression were analyzed using Southern and Northern blots in ten transgenic lines. a Vector map used in this study. LB Left border of T-DNA, RB right border of T-DNA, 35T CaMV 35S terminator, HygR Hygromycin phosphotransferase, 35P CaMV 35S promoter, Nos nos terminator, tflA toxoflavin lyase, EI EcoRI, HIII HindIII, XhI XhoI, KI KpnI, XI XbaI. b Each transgenic line showed various expression levels of transgenes HygR and tflA. Loading was monitored by EtBr staining. c Southern blot of four transgenic lines detected that one to several copies of T-DNA were inserted in each line after digestion of genomic DNA with HindIII. TflA DNA fragment amplified by PCR used as a probe
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T1 progeny of line 28 with multicopies of T-DNA were analyzed to determine the expression level of transgenes HygR and tflA and the arrangement of T-DNAs in the genome (Fig. 2). Because all T1 progeny of line 26 exhibited a high expression level of transgenes, their expression level was used as a control for a high expression level of transgenes. Expression levels more than 2-fold lower compared to this high level were described as low level. Southern blot analysis revealed that all T1 progeny of line 28 germinated on hygromycin containing medium showed the same hybridization pattern as parental T0 line 28 in terms of transgene integration
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Transgene arrangements in the genome
Fig. 2 Unstable expression of transgenes in T1 generation of transgenic plants. a Southern analysis for HygR gene in T1 generations of line 26 and 28. T1 progeny showed the same restriction digestion (HindIII) pattern as parental lines in Fig. 1. b Northern analysis for transgene HygR and tflA in T1 generation of line 28. RNA blot was probed first with tflA gene, reprobed with HygR gene after stripping the tflA probe, and reprobed finally with actin gene as a loading control after stripping
(Figs. 1c, 2a), indicating that several copies of T-DNA were integrated into the same locus or closely linked locus of the genome. All T1 progeny of line 26 also showed the same pattern of Southern blot analysis (Fig. 2a). The expression levels of HygR and tflA genes in T1 progeny of line 28 were different from the T0 plant (Fig. 2b). Only one of five progeny of line 28 showed a similar pattern of transgenes expression to the parental line (Fig. 2b, line 28-1). Interestingly, line 28-3 (one of five progeny analyzed) exhibited almost a high expression level for both transgenes whereas line 28-5 showed the opposite pattern of transgene expression levels, with a lower expression level of HygR and a higher level of tflA than parental line 28, whereas line 28-4 exhibited a lower level of both transgenes (Fig. 2b, line 28). These results suggest that the expression level of transgenes in line 28 became much more unstable in the T1 generation than in the T0 generation.
Transgene arrangements in the genome were analyzed using restriction digestion of genomic DNA of line 26-1 and 28-1 by Southern blot analysis. T-DNA used in this study has several unique restriction sites including EcoRI, HindIII, KpnI, XbaI, and XhoI (Fig. 1a). When tflA gene was used as a probe in line 28 analysis, two bands of approximately 3 and 4 kb were detected in the EcoRI digestion pattern, two bands of greater than 8 and 5 kb in the HindIII digestion pattern, two bands of approximately 6 and 10 kb in the XhoI digestion pattern, two bands of approximately 3 and 2 kb in the KpnI digestion pattern, and several bands including approximately 4, 7, and 10 kb in the XbaI digestion pattern (Fig. 3a). When HygR gene was used as a probe in line 28, more than two bands including approximately 3 and 10 kb in the EcoRI digestion pattern, and greater than 8 and 5 kb in the HindIII digestion pattern were detected (Fig. 3a). The bands including approximately 4 kb in the EcoRI digestion pattern, less than 6 kb in the XhoI digestion pattern, and approximately 3 kb in the KpnI digestion pattern were expected if two T-DNAs were integrated as a repeat in a head-to-head form. All bands in a similar size as expected in a repeat in a headto-head form were detected (Fig. 3). In addition, if two T-DNAs were integrated as a repeat in a tail-to-tail arrangement, approximately 8.4 kb in the HindIII digestion pattern, approximately 6.8 kb in the KpnI digestion pattern, and approximately 4.8 kb in the EcoRI digestion pattern should be detected. Similar bands of 8.4 and 6.8 kb in length were detected as expected (Fig. 3a). However, although a 4.8 kb band was expected to be detected by the HygR gene as a probe, an approximately 3 kb band was detected in the EcoRI digestion pattern. Shortening of the band can be explained based on filler sequences with EcoRI site recombined between two T-DNAs during integration of T-DNA repeat into the genome. In line 26, more than one band in the EcoRI, KpnI, and XbaI digestion patterns was detected (Fig. 3a). The bands detected in line 26 were not found in a repeat arrangement such as a headto-head, tail-to-tail, or tandem arrangement, suggesting that more than one copy of T-DNA might be independently integrated into the genome of line 26 and that the arrangement might not be so complicated as to prevent a high expression of transgenes as in line 28. Usually, the size of T-DNA found in the transgenic plant was not exactly the same as intact T-DNA because some deletion of T-DNA sequence and/or addition of filler sequences of a few to several hundreds of nucleotides would take place during integration of foreign DNA into the genome (Kim et al. 1998; Wang et al. 2005; Windels et al. 2003). Restriction analyses indicated that at least three copies of T-DNA in a head-to-head and tail-to-tail
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Plant Biotechnol Rep Fig. 3 Restriction digestion analysis of genomic DNA for the transgenes and schematic diagram for T-DNA arrangement in line 28. a Southern analyses using restriction enzymes generated restriction digestion patterns to determine the T-DNA arrangements in the genome. HygR or tflA DNA fragments were used as probes. EI EcoRI, HIII HindIII, XhI XhoI, KI KpnI, XI XbaI, 26 transgenic line 26, 28 transgenic line 28-1. Arrows with number indicate the expected DNA length in kb. b Schematic diagram for T-DNA arrangement with repeat structures of head-tohead and tail-to-tail forms in genome of line 28. The restriction sites that give the predicted hybridizing fragments on Southern blots and the sizes (kb) of the predicted fragments are shown. The orientations of the promoters or coding sequences are indicated by arrowed boxes
arrangement were integrated at the same locus of the genome of line 28 (Fig. 3b). However, several bands cannot be explained in this simple arrangement, presuming that T-DNAs were rearranged with another extra copy or different complex copies by the recombination of disrupted T-DNA integrated into the genome. If this is the case, the locus of another single copy or complex copies should be linked closely to the locus in question because no transgene copies were segregated to the T1 progeny of line 28.
Discussion We analyzed the expression level of transgenes and the arrangements of multicopy integration of T-DNA in the genome of transgenic tobacco plants in this study. In line 28, at least three copies of T-DNA were integrated in a complex arrangement of head-to-head and/or tail-to-tail in the same locus or closely linked two loci in the genome (Fig. 3). However, the possibility cannot be excluded that other arrangements including deletion, insertion, and
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translocation of short fragments of T-DNA could also exist (Kim et al. 1998; Wang et al. 2005; Windels et al. 2003). It has been reported that multicopy integration of T-DNA into the genome comparable to line 28 is associated with the gene silencing in transgenic petunia overexpressing chalcone synthase gene (Stam et al. 1997; Van Blokland et al. 1994). A transgene integration in tandem or inverted repeats into the genome is often silenced via RNAi machinery induced by aberrant mRNA transcribed from repeated transgene (Baulcombe 1996; Fojtova´ et al. 2006; Mette et al. 1999). A tail-to-tail form in line 28 seems similar to an inverted repeat in which the transgene is easily silenced because antisense RNA can be expressed via read-through transcription. A head-to-head form of multicopy integration of transgene could overexpress the transgene compared to a single copy integration; however, it could simultaneously trigger to induce gene silencing because of aberrant mRNA transcription (Romano and Macino 1992; Susi et al. 2004). Therefore, we expected that HygR and tflA genes in line 28 would be silenced because of a complex arrangement of T-DNA in the
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genome (Fig. 3b). However, the expression level of transgenes was kept unstable and more varied in the T1 generation than in the T0 generation, even though all T1 progeny were maintained in similar growth conditions. In vitro culture conditions and temperature often affect transgene expression and silencing (Fojtova´ et al. 2003; Meza et al. 2001). Although being expressed transgenic plants at initial stages, transgenes including GUS, nptII, chitinase, b-1,3glucanase, luciferase become silenced in the second or third generation in Arabidopsis and wheat (Anand et al. 2003; Meza et al. 2001), suggesting that transgene silencing is intensified through successive generations. Furthermore, it has been reported that transgene is silenced when multicopies of T-DNA are integrated in an arrangement of tandem repeat or inverted repeat at the same locus of the plant genome (Chan et al. 2006; De Buck et al. 1999; Martienssen 2003; Muskens et al. 2000). However, as demonstrated in this study, multicopies of transgenes integrated in a complex arrangement at the same locus exhibited various expression levels in each T0 plant, and even in each individual plant of the progeny of a particular transgenic plant transgenes expressed in different levels, which is against our prediction that all progeny would show an equivalent level of transgene silencing. Therefore, we conclude that transgene silencing is governed in a more complicated manner than previously explained. Acknowledgments This work was supported by a grant (Code 20070301034020) to W.J.J. from BioGreen 21 Program funded by the Rural Development Administration, a grant to J.R.L. from the Crop Functional Genomics Center of the 21st Century Frontier Research Program, a grant to J.R.L. from the Korea Science and Engineering Foundation through the Plant Metabolism Research Center of the Kyung Hee University funded by the Korea Ministry of Education, Science and Technology, and a grant to J.R.L. from the Marine Extreme Genome Research Center funded by the Korean Ministry of Marine Affairs and Fisheries.
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