Microsoft Word - Template_Authors_arjournals_Biol-1.doc Challenge towards plant recombinant protein expression: instability in nuclear and chloroplast transformation   Mahshid Amiri, Mokhtar Jalali-Javaran*, Parastoo Ehsani, Raheem Haddad All Res. J. Biol., 2016, 7, 13-19 The publication cost of this article might be covered by external sponsors. More info for sponsors at: sponsors@arjournals.com ARTICLE                                                                                                                    Issue 1, Vol 7, 2016, 13-19 Challenge towards plant recombinant protein expression: instability in nuclear and chloroplast transformation   Mahshid Amiri1, Mokhtar Jalali-Javaran*1, Parastoo Ehsani2, Raheem Haddad3 1  Department of Plant Breeding & Biotechnology, Faculty of Agriculture, Tarbiat Modares University(TMU), Tehran, Iran; Molecular Biology Department, Pasteur Institute of Iran, Tehran, Iran Department of Agricultural Biotechnology, Imam Khomeini International University, Qazvin, Iran Corresponding author: Mokhtar Jalali-Javaran: Department of Plant Breeding & Biotechnology, Faculty of Agriculture, Tarbiat Modares University(TMU), Tehran, Iran; jalali.mokhtar@gmail.com,Tel: 098-2148292104       Grafical Abstract         Abstract: It is crucial to maintain the stability of transgene and its expression level. It seems the transformation method and the target organ can influence this instability. To this aim, two transformation systems, Agrobacterium-mediated and particle bombardment systems which have been applied to introduce tissue plasminogen activator (tPA) into nuclear and chloroplast respectively, have been compared to determine transformation efficiency, tPA expression, and stability. The presence of the tPA gene in transformants has been confirmed by PCR analysis. The gene expression in nuclear transformants and homoplasmy in transplastomic plants have been assayed by ELISA and southern blot analyses, respectively. Some of the Agrobacterium- derived transformants have shown the heritability and stability of the integrated T-DNA harboring the transgene, which encodes the tissue plasminogen activator and instability of its expression in the T1 generation. Using Southern blot analysis of bombardment-mediated transformants has surprisingly led to detecting the inheritability of tPA. There are several factors lead to the silencing of in transgenic plants, which should be considered. Possible reasons for these silencing are likely vector designing, methylation, copy number, and genome rearrangement. Keywords: Recombinant Protein, Tissue plasminogen activator, Agrobacterum, Particle Bombardment, Instability   Introduction The pharmaceutical industry has needed the production of recombinant proteins in sufficient quantity in order to keep up with demands 1, 2. There are other factors, besides the quantity, which can be obtained by recombinant technology such as increased quality, enhanced safety, and decreased cost 3. Some living cells have been engineered as heterologous expression platforms to produce recombinant protein, including bacteria, animals, and plants 4. Higher Gene Transformation system Agrobacterium- mediated Particle bombardment T1 generation I n s t a b i l i t y T1 generation Vector designing Methylation Copy number Genome rearrangement 13 All Res. J.Biol, 2016, 7, 13-19     plants offer some advantages 4 which emphasize the practicality of utilizing them as green factories to express useful recombinant protein 5: lowering costs of production, no human pathogens, synthesizing protein with correct folding, and post-translational modification like glycosylation 6.   Considering these advantages, up to date, several biopharmaceutical proteins have been produced in the plants 7, so, plant molecular farming has impacted positively on the important pharmaceuticals 8. Plant molecular farming has enrolled genetically engineered plants as vehicles for expression of recombinant protein and provided an attractive perspective to produce these important proteins in a large scale at low costs 9.   This plant engineering refers to the introduction and integration of "interested" DNA in plant cells, which can lead to transient or stable expression of interested DNA 10. Through either nuclear or plastid genomes, stable- transformed plants can be obtained 11, although, chloroplast transformation offers several advantages in comparison with nuclear transformation such as minimizing or avoiding the gene escape, eliminating the position effect, and higher expression level 12. As a consequence of stable transformation, the interested DNA is integrated into the host DNA and eligibly predicted to be passed on to the next generation 10. After about two decades, in which molecular farming is coming of age 7, the concentration has moved away from technical and principal studies towards a serious attention of necessity for sustainable production of recombinant protein 9.   In fact, it can be remarked that the stability is as important as the expression level of transgene for the large-scale commercialization of transformants 13, it is clear that the expression and stability are not guaranteed over generations 14. Transgene instability can be defined as the loss of a transgene or its expression in genetically engineered organisms15; methylation, genome rearrangement, and the site of insertion found to be responsible for the phenomenon 16. The instability has been reported in both biolistic bombardment and Agrobacterium mediation 17.   Tissue plasminogen activator (tPA), a serine protease, hydrolyses the plasminogen to convert it to plasmin 18. tPA and plasminogen bind to the fibrinogen and fibrin to modulate proteolytic activity, enable the dissolution of blood clots. It has been found that it is useful to treat myocardial Infarction, thrombosis, and stroke 19. It should be noted that there is a single-chain nonglycosylated form of tPA called reteplase, with a longer plasma half-life, better diffusion, and higher fibrinolytic activity 20. As utilizing tPA one hour after heart attack can lead to increasing the survival chance, there has been an interest to produce tPA in large scale 21. Attempts have been made to produce the protein in several expression systems like Saccharomyces cerevisiae 22, Aspergillus nidulans 23, Escherichia coli 24, Chinese hamster ovary (CHO) cells 25, Leishmania 26, Bowes melanoma cell line 27, mammalian cell lines 28, 29,and insect cells 30, however the aforementioned disadvantages lead to recent interests in plants. It has been expressed in tobacco 21, 31, 32 and oriental melon 33.   In this study, we compared the stability and inheritance of two forms of transgene in transformants transformed by different methods (Agrobacterium-mediated transformation and particle bombardment technique) in different organs (nuclear and chloroplast, respectively). In fact, transgenic plants were investigated to determine the transgene and expression stability over generations because the probability of instability/ transgene silencing, through generations, increases 34. Our attempts were made to find the stable transgenic plants and discuss the stability and expression level of transgene.     Methods and materials   Seed culture   Two groups of transgenic plants were considered to assay. Nicotiana tabacum CV. Xhanti were transformed by the Agrobacterium-mediated method and particle bombardment technique.   Nuclear primary transformants (T0) created through Agrobacterium-mediated transformation harboring pBIt-PA construct containing the Kozak sequence before the start codon and a KDEL sequence before the stop codon, nptII gene was controlled by 35S promoter of the cauliflower mosaic virus (CaMV) and NOS terminator of the Agrobacterium tumefaciens 32. The seeds of T0 were cultivated to obtain T1 seeds. pKCZK2S carrying K2S and aadA gene controlled by Prrn as the rRNA operon promoter and rbcl3ʹ′chl of the Chlamydomonas rbcl gene as a terminator was applied to make chloroplast transgenic plants. After several selection rounds and four rounds of regeneration, plants were allowed to produce T1 seeds 21. All seeds were grown in the pots containing a homogeneous mixture of perlite and peat moss (1:3 ratio of perlite:peat moss).   PCR analysis   Genomic DNA was extracted from fresh leaves of transgenic and non-transgenic plants utilizing the CTAB method 35 and used as the template in the PCR assays. PCR was conducted to confirm the presence of both tPA and K2S genes (a truncated form of tPA) in the nuclear and chloroplast genome, respectively. Two primer sets were used on each DNA template: (1) one to amplify a 1.7 kb tPA (forward: 5ʹ′- GAGTCTAGATAAACAATGGATGCAATGAAGAGAGG G-3ʹ′; reverse: 5ʹ′- ATAGTCAACTCATAGCTCATCTTTCGGTCGCATGTT G-3ʹ′) and (2) another to amplify a 1.2kb K2S (forward: 5´- GGAAACAGTGACTGCTACTTTGGGAATGG-3´ and revers 5´- TCACGGTCGCATGTTGTCACGAATCCAG- 3´).   Southern blot   14 All Res. J.Biol, 2016, 7, 13-19     To confirm the homoplasmy, a Southern Blot analysis was conducted on chloroplast transgenic plants. High-quality genomic DNA was isolated from the fresh leaves of T1 PCR- positive plants by the CTAB method 36. High quality genomic DNA of transgenic and non-transgenic plants were digested by HindIII, fractionated at 20V through a 1% agarose gel for 16 h, and transferred onto nitrocellulose membranes (BioRad, USA) utilizing a traditional wet system. Fragments designed based on a flank region in the chloroplast genome, specifically amplified using the DIG- DNA Labeling and Detection kit (Roche, Germany) and the primer set (P-Forward 5´- ATGTGTAATGATTCCCCCATTC-3´ and P-Reverse 5´- CTTCTCTCCCACTTCACACCTC-3´), were utilized as hybridization probes, producing a 1kb size fragment in non- transgenic(non-transplastomic) plants and a 2.8 kb in transgenic plants (transplastomic) along with a positive control(pKCZK2S vector). Based on the manufacturer’s protocol, the probe was hybridized and resolved on the membrane.   ELISA   The level of specific tPA in leaves of T1 PCR-positive transgenic plants was estimated by enzyme-linked immunosorbent assay (ELISA) on protein extracted just before the assay. tPA was assayed by an indirect ELISA procedure as previously described 21. Result   Analysis of transformed plants by PCR   PCR was carried out with two primers sets to confirm the presence of recombinant tPA gene in transformants. The presence of 1.7 kb in some nuclear transformed plants, about 20 plants out of 200 plants, has showed the inherited integration of the transgene in these plants (Fig.1). DNA from some chloroplast transformed plants has represented the expected size of amplified product to be 1.2 kb with K2S- gene-specific primers and some of them have shown no band.(Fig.2). Untransformed plants (negative control) have shown no PCR product.   Figure  1 PCR  amplification  of  a  1.7  kb  fragment.  M:  1  kb  molecular  weight   marker   (Fermentas),   C-­‐:   negative   control,   C+:   positive   control   (pBIt-­‐PA   vector   contain   tPA   gene),   Wt:   non-­‐transformed   plant,   1-­‐6:   transformed   plants.     Figure  2 PCR  amplification  of  a  1.2  kb  fragment.  M:  1  kb  molecular  weight   marker   (Fermentas),   C-­‐:   negative   control,   C+:   positive   control   (pKCZK2S   vector  contain  K2S  gene),  Wt:  non-­‐transplastomic  plant,  1-­‐5:  transplastomic   plants.   Southern blot analysis   After confirming the presence of interested gene K2S in the chloroplast transformed plants by PCR, it was subjected to the southern blot analysis. As it was expected to observe just a 2.8 kb band on the membrane revealing homoplastic plants and a 1kb size band on the me mbrane indicating a heteroplasmic plant, we just observed the 1kb size band in all of our T1 PCR-positive plants (Fig.3). Our analyses which represented the heteroplasmic plants were not suitable for more analysis like ELISA.   ELISA   An ELISA was conducted to quantify protein by comparing the absorbance readings of the three replicates of T1 PCR- positive plants of nuclear transformation with known quantities of the commercial tPA protein (Alteplase). After estimating, it was found there was not any difference between transgenic and non-transgenic plants in tPA protein content (Data was not shown).   Discussion   As it can be observed in the Fig.1 and Fig.2 the positive-PCR transgenic plants have been obtained in both nuclear and chloroplast transformations, about 10% plants in nuclear and, surprisingly, some chloroplast transformants. Considering the advantages of chloroplast genetic engineering over nuclear ones, which was mentioned above; due to the lack of gene silencing 37; it is assumed to see all chloroplast transformants represent the expected 1.2 kb band. So, by this experiment, we can observe the genomic instability of chloroplast transgenic plants causing the loss of the transgene. To assess the homoplasty, the Southern blot was conducted on the positive-PCR transgenic plants transformed by particle bombardment. Plants show only the expected size for the wild-type plastom band while no integrated transgene bands can be observed. It seems this lack of the 2.8 kb transgene band is precise and needs to be considered.   It can be inferred this phenomenon is the consequence of the intramolecular recombination event resulting in keeping the wild-type plastome copies during the chloroplast segregation procedure 38. To find out more about this phenomenon, a detailed study has been made on the cloning procedure and 15 All Res. J.Biol, 2016, 7, 13-19     the vector structure. pKCZK2S vector was used to create transgenic plants producing tPA. The problem faced in plants transformed by pKCZ has been the same in their next generation 38–40; plants have never met the genetically uniform lines, and comes back to the intramolecular recombination via the repeated sequence element, especially regulatory elements controlling foreign gene expression. As it is visible in the Zou (2003) study, there are three copies of plastid promoter prrn governing both selection markers (aadA) and reporter gene(uidA), two prrn copies are oriented as the direct repeats; specific to transplastomic aadA and endogenous rrn16 (16S rRNA) genes. The result of intramolecular recombination of these direct repeats of prrn leads to the removal of ~10kb including all rRNA (5S, 4.5S, 23S and 16S), the whole uidA cassette, and several tRNA genes and keeps the aadA cassette resulting in aberrant transplastomes  which are spectinomycin resistant.   Figure   3 Sothern   blot   analysis   of   transplastomic   T1   Plants   with   Hind   III   enzyme.   M:   1   kb   molecular   weight   marker   (Fermentas),   Wt:   non-­‐ transplastomic  plant,  C+:  positive  control  (pKCZ  vector  contain  K2S  gene),  1-­‐ 5:  transplastomic  plants.   Abdoli-Nasab et al (2013) decided to add the tPA cassette before aadA one to overcome this problem, so, in their opinion, transplastomes could express both tPA and aadA simultaneously in spectinomycin resistant plants. They remained unaware that the removal of all rRNA and several tRNA genes were lethal for transplastomes, however aadA was expressed resulting in the resistance to the spectinomycin. In their analysis, on the one hand, they did not address the issue as they analyzed transformants in the early stages of growth in T0 generation. The average of transgene loss was estimated about 5%, but it was a continuous process and did not stop until all intramolecular recombination via the direct repeats happened, resulting in all rRNA and several tRNA genes to be removed 41. On the other hand, the intramolecular recombination procedure under selection stress seemed to be a slow process, but accelerated in the absence of antibiotics like growing in the soil, so, it seemed to be a plausible interpretation as to why we met the loss of transgene in the T1.   Intramolecular recombination via direct repeats is not always a potentially lethal adverse phenomenon in transformation; this strategy has been helpfully used to create antibiotic marker-free transplastomic plants, 41 while one of the direct repeats has been left in the chloroplast genome after the recombination.   To applicate transformation technologies commercially, it is extremely important that transformants express the recombinant protein over generations 42. The evaluation of genetic instability seems to be obvious via a loss or sudden change in the phenotype of agronomically engineered plants 43 while more studies need to be considered in non- agronomically engineered ones. In our study, as shown in Fig.1, we have found 20 plants out of 200 plants have shown positive in PCR. These PCR-positive plants have not represented any difference among transgenic and non- transgenic plants in tPA protein content.   The transgene instability can increase over generations especially when it advances to stabilize plants in the homozygous level 44.The instability of transgene inheritance, in the form of transgene lost or rearrangement, has been reported in the PEG-mediated direct gene transfer, bilolistic bombardment or Agrobacterium mediation 17. Although Agrobacterium-mediated transformation shows less rearrangement, co-suppression and instability in subsequent generations in comparison with bombardment transformation 44. There are several reasons which should be considered as the answer for this event; several factors are generally accepted to be responsible for transgene expression instability such as methylation, copy number, genome rearrangement, integration site in genome, and endogenous gene homology to the transgene 16.   It is crucial to produce single-copy number inserted transformants, because it is known that the loss of transgene expression and the expected phenotype can also result from multiple transgene copy numbers 45, 46. In addition, the tandem copies of T-DNA are often transferred at a single locus in the Agrobacterium-mediated transformation and it is found that the T-DNA repeats which stand inverted and head- to-head around the right border are often associated with transgene silencing 47.   Epigenetic interactions as another potential source of transgene instability have been known as gene silencing system in plants 48, resulting from the interaction among multiple transgene copy number or an endogenous gene homology to the transgene leads to the homology dependent gene silencing (HDGS). The mechanism has been unclear, although some theoretical hypothesis have put forward that HDGS may be observed either through the transcription repression, called transcriptional gene silencing (TGS), or through mRNA degradation, called posttranscriptional gene silencing (PTGS) 49. TGS is characterized as heavy methylation to be the mechanism of the silenced promoter and is meiotically heritable 50, it can be interpreted that the chromatin environment of the transgene is epigenetically modified in a way that can be heritable, most likely before or during gametogenesis. In fact, promoter regions in the repeated transgenes are often methylated, these repeated transgenes cause the methylation of homologous regions placed in trans; also transcription of aberrant promoters shows promoter methylation. Heavy methylation can be seen 16 All Res. J.Biol, 2016, 7, 13-19     when prokaryotic vector sequences integrate into the plant genome, the excess vector sequences cannot be well tolerated by the eukaryotic genome and are often spontaneously resulted in heavy methylation that can extend into neighboring transgenes 49 it is possible these sequences have unusual compositions to bind the eukaryotic nuclear proteins, which subsequently are susceptible to plant methyltransferases 49 and to a conversion to different epigenetic states 50. PTGS is not meiotically inheritable and should be reinstituted in each gametogenesis. It leads to methylation of an increased amount of DNA within the protein-coding region, resulting in the appearance of specific low molecular weight fragments of RNA 49.   Studies reported that the heritable instability could occur in the T2 to T4 generations, although Travella et al (2005) reported that there was some evidence in which transgene instability occasionally could be appeared in the T1 population which contained the single-copy number of interested gene, as mentioned above the extra integrated vector sequence led to gene silencing, however it was recently reported that this extra vector sequence might influence transgene expression 51. Sometimes the meiotic instability results in the loss of T-DNA partially or completely, this instability frequently has been reported to be low for Agrobacterium-mediated transformants with single- copy inserts 17.   Considering the aftermentioned reasons for the problem which we have met in the Agrobacterium-mediated transformants in the T1 generation, according to Scott et al (1998), it seems it is early to discuss the definite answer for this event now and more experiments require to be assayed. Using scaffold attachment regions (SAR) can be useful in nuclear stable transformation, as SARs can be attached to the proteins of the nuclear scaffold and can lead to the organization of the chromatin into loop exposure to transcription machinery. So, flanking the transgenes with SAR elements have been shown to contribute to the reduction of position effects and silencing of transgene expression 52– 54.     References     1 Joshi, L., and Lopez, L.C. (2005). 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