Abstract:
An engineered plant-derived transfer (P-)DNA was designed and constructed based on a couple of T-DNA homologous sequences in sugar beet genome. Plant transformation efficacy of the engineered P-DNA was analyzed compared to conventional T-DNA in two independent systems, stable transformation of tobacco plant and sugar beet hairy roots. The outcomes demonstrated that plant transformation is directed well by vectors carrying the engineered P-DNA with higher efficiency than the conventional binary vector in both experimental systems. This vector was further improved by adding two matrix attachment regions within P-DNA. This new vector was even more efficient in gene transfer, higher than conventional binary vector.

Description:
CROSS REFERENCE TO RELATED APPLICATION 
       [0001]    This application claims the benefit of priority to an Iran Application Serial Number 139450140003008184 filed on Oct. 20, 2015, which is incorporated by reference herein in its entirety. 
       TECHNICAL FIELD 
       [0002]    The present disclosure relates generally to the production of binary vectors with high transformation rates of any organism and more specifically plant species, that carry an engineered P-DNA sequence derived from sugar beet genome. 
       BACKGROUND 
       [0003]    New transgenic crops with improved farming, food and feeding traits are progressively being developed. Although the commercialization of transgenic plants started early 1990s, necessity for analysis of unintended effects of the transgenes hampers preparing required documents for their release. Currently,  Agrobacterium -mediated plant genetic engineering methods relies on the integration of foreign (often prokaryotic) DNA, such as transgenes, promoters, terminators and T-DNA, into plant genomes. For example, cry3 gene (GenBank Accession AY572010.1), coding for a BT protein, contains 2.7% of CpG motif, a known characteristic of prokaryotic DNA). Some interactions between inserted prokaryotic DNA and plant immune system are documented. 
       SUMMARY 
       [0004]    The present application discloses a series of novel binary vectors carrying an engineered sequence that include P-DNA left and right borders (LB and RB) derived from two loci in sugar beet genome. The vectors are further comprised of modified RB flanking sequences including an upstream AC-rich region (ACR) and downstream of right border (DR), upstream of left border (UL), LB downstream sequences including a series of C-clusters and two copies of Rb7 matrix attachment regions (MAR). The effectiveness of such vectors was verified by two transformation systems, transgenic tobacco plants and sugar beet hairy roots. The transformation efficiency of the vectors was compared with a similar binary vector carrying  Agrobacterium  T-DNA, pART27. 
         [0005]    To examine P-DNA functionality, it was substituted for conventional T-DNA in SalI restriction sites of pART27 binary vector which is a derivative of pTiC58 carrying elements such as RB, LB, ACR and C-clusters. An expression cassette carrying Pnos-nptII-Tnos encoding neomycin phosphotransferase (nptII) was also cloned between the borders of P-DNA in the ClaI restriction site of pAPB to produce pAPn vector ( FIG. 3 ). In a sugar beet hairy root system, transformation efficiencies of the vectors were compared with a similar binary vector carrying  Agrobacterium  T-DNA, pART27. Transformation rate was defined as the PCR-positive percentage of hairy roots per leaf explants. 
         [0006]    P-DNA ability to integration of transgene into the plant cell genome was determined in comparison with conventional T-DNA. Independent transgenic tobacco plants carrying either P-DNA in pAPn or T-DNA in pART27 containing nptII expression cassette were examined. Here, transformation rate was defined as the PCR-positive percentage of regenerated plants per leaf explants. Transformation rate was increased to as high as four fold by the use of the new binary vectors. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0007]    Features of the subject technology are set forth in the appended claims. However, for purpose of explanation, several implementations of the subject technology are set forth in the following figures 
           [0008]      FIG. 1  illustrates the map of the synthesized plant-derived DNA, according to one implementation of the instant application 
           [0009]      FIG. 2  illustrates the comparison between the free energy of P-DNA and T-DNA 
           [0010]      FIG. 3  illustrates the cloning strategy for producing pAPB and subsequently pAPn vectors 
           [0011]      FIG. 4  illustrates the map of pAPn binary vector 
           [0012]      FIG. 5  illustrates the cloning strategy for producing pAPF2rn vector 
           [0013]      FIG. 6  illustrates the map of pAPF2rn vector 
       
    
    
     DETAILED DESCRIPTION 
       [0014]    In the following detailed description, various examples are presented to provide a thorough understanding of inventive concepts, and various aspects thereof that are set forth by this disclosure. However, upon reading the present disclosure, it may become apparent to persons of skill that various inventive concepts and aspects thereof may be practiced without one or more details shown in the examples. In other instances, well known procedures, operations and materials have been described at a relatively high-level, without detail, to avoid unnecessarily obscuring description of inventive concepts and aspects thereof. 
         [0015]    Considering high cost of transformation and then risk assessment experiments for transgenic plants, needed for their release and commercialization, it is necessary to take possible means to increase transformation rate and to reduce required steps for risks analysis. Using P-DNA instead of conventional T-DNA in binary vectors for  Agrobacterium -mediated plant transformation is a key feature of minimizing the presence of prokaryotic DNA in transgenic plants. In this application, a new engineered P-DNA derived from sugar beet genome was designed and successfully used for transformation of plants in two independent systems including stable transformation of tobacco and hairy roots of sugar beet. Such P-DNA fragments derived from petunia, potato,  Arabidopsis thaliana  and  Medica gotruncatula  have already been recognized by bioinformatics analysis. The potato P-DNA was used for stable plant transformation. However, the novel engineered P-DNA elements with modified UL and DR sequences and MAR sequences in the context of binary vector presented in this application is advantageous for increasing the transformation rate as well as stability of transgene expression. Plant transformation efficacy of sugar beet derived P-DNA was compared with that of conventional T-DNA. Results showed transformation efficiency of P-DNA was 2.3 and 2.2 fold higher than that of a conventional T-DNA binary vector in tobacco plants and sugar beet hairy root systems, respectively. Transformation was further increased twice by the use of MAR sequences in the context of P-DNA. 
         [0016]      FIG. 1  shows the sequence of the engineered P-DNA derived from two loci in sugar beet genome. A 283-bp region from RB to LB (Green boxes) consists of a 208-bp sequence of RB-like (GenBank: FR852871.1) and a 75-bp sequence of LB-like (GenBank: FR852867.1) parts of sugar beet genome. A little pink box indicated the first base of LB-like part. Some elements like DR and UL were indicated by yellow boxes. ACR region and C-clusters of  Agrobacterium  pTiC58 were shown by little blue boxes. 
         [0017]    In  Agrobacterium -mediated plant transformation methods, a cleavage within the 25-bp right border (RB) sequence by a virD1/D2 protein complex is needed for initiation of T-DNA transfer. Also, RB flanking sequences including an ACR and a DR decamer influence the efficacy of T-DNA transfer initiation. The second cleavage for the transferring T-DNA also requires the presence of a 25-bp LB sequence, an AT-rich motif with a consensus sequence positioned at UL as well as LB downstream sequences including a series of C-clusters. Search in publicly available sugar beet genome sequence led to identification of an RB-like (GenBank Accession FR852871.1) and an LB-like (GenBank Accession FR852867.1) sequence with maximum identity to the known T-DNA borders and relevant elements including DR and UL. To design an optimal P-DNA in the inventive binary vectors, a 208-bp sequence of RB-like was fused with a 155-bp of LB-like sequence. Some single nucleotide substitutions for optimization of the essential elements roles by considering the consensus sequences or for making some enzymatic restriction sites for following cloning steps were included. ACR region and C-clusters of  Agrobacterium  pTiC58 vector were placed just before RB and after LB sequences, respectively. 
         [0018]    As used throughout this application, unless otherwise indicated Sequence ID 1 (“SEQID1”) refers to the sequence of the P-DNA shown in  FIG. 1 . Specifically, in this application SEQID1 means 
         [0000]    
       
         
               
             
           
               
                 GTCGACCCGAGGGGAACCCTGTGGTTGGCATGCACATACAAATGGAC 
               
               
                   
               
               
                 GAACGGATAAACCTTTTCACGCCCTTTTAAATATCCGATTATTCTAA 
               
               
                   
               
               
                 TAAACGCTCTTTTCTCTTAGGTTTACTATGGAATATATCCTGGGTGT 
               
               
                   
               
               
                 GGCCGAAGTCTGTTCGGGAAGCTTTAATTCAATGGGTTTACCCAAAG 
               
               
                   
               
               
                 AAAGGAAAATTCTTCAAACAAATTTGAGCTCTGCATCTTTTCAAGGT 
               
               
                   
               
               
                 ACCATTATATGGTCAGTGTGCTCGAGAGAGAGAAATGAATCTAGAAT 
               
               
                   
               
               
                 TTTCAAGCGGCCGCAACAAATATCGATTCGCCTCTGGAAAAGGGAGG 
               
               
                   
               
               
                 ACGTGCTATATTGATTTTTGATTCACTCTCAAGCTTGCCCTTATATT 
               
               
                   
               
               
                 ACATGTCGATATATCCTGCCCAAGCTTCCAGCCAGCCAACAGCTCCC 
               
               
                   
               
               
                 CGACCAGATCTGTCGAC. 
               
             
          
         
       
     
         [0019]      FIG. 2  shows analysis of free energy (Kcalmol −1 ) by WEB-THERMODYN across RB and its 200-bp upstream sequence of P-DNA in comparison with T-DNA. The analysis shows that despite the similarity of P- and T-DNA free energy profile, the values calculated for P-DNA were higher than those of T-DNA (149.39 versus 118.21, respectively). It leads to the lower helical stability of P-DNA RB region, which subsequently may facilitate the first cleavage of P-DNA and initiation of P-DNA transfer. 
       Example 1 
       [0020]    Construction of pAPn and pAPF2rn Vectors 
         [0021]      FIG. 1  illustrates the cloning strategy for producing pAPB followed by cloning strategy for producing pAPn constructs. A 283-bp and a 155-bp sequences corresponding to RB and LB of P-DNA, respectively, were selected from sugar beet genome deliberately such that only the essential elements for DNA transfer were included. Moreover, it contains a high A/T content (59.4% versus 51.1% for T-DNA), which may affect initiation of the P-DNA transfer, as well. As illustrated in  FIG. 3 , pAPB vector was constructed in several steps through conducting conventional digestion and ligation reactions. In fact, the synthesized P-DNA substituted a conventional T-DNA between SalI restriction sites of pART27 binary vector, which is a derivative of pTiC58 carrying elements such as RB, LB, ACR and C-clusters. To examine P-DNA functionality, an expression cassette carrying Pnos-nptII-Tnos encoding neomycin phosphotransferase (nptII) was also cloned between the borders of P-DNA in the ClaI restriction site of pAPB to produce pAPn vector  FIG. 4 .  FIG. 4  illustrates a map of pAPn binary vector. Restriction sites and an example of gene expression cassette for nptII gene are shown. The synthesized PB sequence substituted a conventional T-DNA between SalI restriction sites of pART27 binary vector, which is a derivative of pTiC58 carrying elements such as RB, LB, ACR and C-clusters. Furthermore, two copies of Rb7 matrix attachment regions (MAR) were placed after RB and before LB leading to construct pAPF2rn vector as shown in  FIGS. 5 and 6 . Specifically,  FIG. 5  illustrates a cloning strategy for producing pAPF2rn vector starting from pAPn construct.  FIG. 6  illustrates a map of pAPF2rn binary vector. Restriction sites are as shown in  FIGS. 4 and 5 . An example of gene expression cassette for codA gene is shown in  FIG. 6   
       Example 2 
       [0022]    Increasing Transformation Rate of Tobacco Cells Using pAPn Vector 
         [0023]    Leaf explants of  Nicotiana tabacum  L. cv Xanthi were transformed with  Agrobacterium tumefaciens  strain GV3101 carrying pAPn, the newly developed binary vector containing either P-DNA or pART27, as a conventional binary vector containing T-DNA. Overnight-grown bacterial cultures were precipitated for 10 min at 2800×g and re-suspended in 50 ml LB medium containing 100 μM acetosyringone to 0.5 OD 600 . These cultures were used to infect leaf and petiole segments of tobacco for 10 min. The infected segments were incubated for 72 h on co-culture medium (MS medium supplemented with 400 mg lit −1  cefotaxime, 50 mg lit −1  kanamycin and 0.1% agarose plus 1 mg lit −1  BA and 0.1 mg lit −1  IBA) at 25° C. in dark. They subsequently transferred to regeneration medium (MS medium supplemented with 1 mg lit −1  BA, 0.1 mg lit −1  IBA, 300 mg lit −1  cefotaxime, 100 mg lit −1  kanamycin and 0.1% agarose). They were sub-cultured in two week intervals. After two months regenerated shoots were isolated and placed on hormone free growth medium (MS medium supplemented with 300 mg lit −1  cefotaxime, 100 mg lit −1  kanamycin and 0.1% agarose). Transformed plants were monitored by performing a PCR using Pnos-F (5′-GAATTCGGCCGGGAGCATGCGAC-3′) and nptII-R (5′-TGATCATTTCGAACCCC AGAGTC-3′) primer pairs. 
         [0024]    As shown in Table 1, the transformation efficiency of pAPn was significantly greater than that of pART27 at P&lt;0.05. These results indicate that the transformation efficiency using pAPn vector was 2.3 fold higher than when pART27 vector was used. 
         [0000]    
       
         
               
             
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 The rates of produced transgenic tobacco shoots 
               
               
                 using either pAPn or pART27 binary vectors 
               
             
          
           
               
                   
                   
                   
                 Kanamycin 
                   
                 No. of 
                   
                   
                   
               
               
                   
                 Transfer 
                   
                 resistant 
                   
                 Examined 
                   
                 PCR +   
               
               
                 Constructs 
                 DNA 
                 Explant 
                 regenerated 
                 Regeneration 
                 plants with 
                 PCR +   
                 Plants 
                 Transformation 
               
               
                 Name 
                 type 
                 No. 
                 plants 
                 rate 1   
                 PCR 
                 plants 
                 rate 2   
                 rates 3   
               
               
                   
               
             
          
           
               
                 pART27 
                 T-DNA 
                 136 
                 45 
                 33% 
                 45 
                 35 
                 77.8% 
                 26% 
               
               
                 pAPn 
                 P-DNA 
                 302 
                 234 
                 77% 
                 74 
                 59 
                 79.7% 
                 61% 
               
               
                   
               
               
                   1 The regeneration rate is the frequency of independent kanamycin-resistant regenerated buds per total number of explants multiplied by 100. 
               
               
                   2 The PCR positive rate is the number of PCR positive regenerated plants per total number of examined plants multiplied by 100. 
               
               
                   3 Transformation rate is PCR positive percentage multiplied by regeneration rate. 
               
             
          
         
       
     
       Example 3 
       [0025]    Producing Sugar Beet Hairy Root Using pAPn Vector 
         [0026]    For hairy root transformation, leaves and petioles of sugar beet variety SBSI-02 were exposed to  Agrobacterium rhizogenes  strain 15834 carrying either pAPn or pART 27 vectors. Overnight-grown bacterial cultures were precipitated for 10 min at 2800×g before re-suspension in 50 ml LB medium containing 100 μM acetosyringone to OD 600  of 0.5. The infected leaf and petiole segments of sugar beet were incubated for 48 h on co-culture medium (MS supplemented with 100 μM acetosyringon) at 25° C. in dark. They were subsequently transferred to rooting medium (MS supplemented with 400 mg lit −1  cefotaxime, 50 mg lit −1  kanamycin and 0.1% agarose) and subcultured every two weeks. After 1 month roots were separated and grown in liquid medium (MS supplemented with 400 mg lit −1  cefotaxime and 50 mg lit −1  kanamycin) while shaking at 100 rpm for two months. Formations of hairy roots were traced by performing a PCR using Pnos-F and nptII-R primer pairs as above. 
         [0000]    
       
         
               
             
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 2 
               
             
             
               
                   
               
               
                 Increased rate of hairy root formations using pAPn vector versus 
               
               
                 pART27 vector 
               
             
          
           
               
                   
                   
                   
                   
                   
                 Examined 
                   
                   
                   
               
               
                   
                 Transfer 
                   
                 Kanamycin 
                   
                 root 
                   
                 PCR +   
               
               
                 Constructs 
                 DNA 
                 Explant 
                 resistant 
                 Rooting 
                 No. with 
                 PCR +   
                 roots 
                 Transformation 
               
               
                 Name 
                 type 
                 No. 
                 roots 
                 rate 1   
                 PCR 
                 roots 
                 percentage 2   
                 rates 3   
               
               
                   
               
             
          
           
               
                 pART27 
                 T-DNA 
                 36 
                 105 
                 292% 
                 25 
                 20 
                 80 
                 234% 
               
               
                 pAPn 
                 P-DNA 
                 39 
                 205 
                 525% 
                 22 
                 22 
                 100 
                 525% 
               
               
                   
               
               
                   1 The rooting rate is the frequency of independent kanamycin-resistant regenerated hairy roots per total number of explants multiplied by 100. 
               
               
                   2 The PCR positive percentage is the number of PCR positive hairy roots per number of examined plants multiplied by 100. 
               
               
                   3 Transformation rate is PCR positive percentage multiplied by rooting rate. 
               
             
          
         
       
     
         [0027]    As shown in Table 2, the frequency of explants producing kanamycin-resistant hairy roots was an average number of 5.2 hairy roots per inoculated explants for pAPn and an average number of 2.9 for pART27. Transformation rate was defined as the PCR-positive percentage of hairy roots per leaf explants. The transformation rate of pAPn was 525% which was significantly (P&lt;0.05) higher than that of paRT27 at 234%. As a result, the transformation efficiency of pAPn was 2.2 fold greater than that of pART27 in sugar beet hairy root system. In other words, the engineered sugar beet derived P-DNA used in pAPn binary vector supports more effective transfer of transgene into plant cells than the conventional T-DNA. 
         [0000]    
       
         
               
             
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 3 
               
             
             
               
                   
               
               
                 Increased rate of hairy root formations using pAPn or pAPFrn versus 
               
               
                 pART27 vectors. 
               
             
          
           
               
                   
                 Transfer 
                   
                   
                   
                   
                   
                 PCR +   
                   
               
               
                 Constructs 
                 DNA 
                 Explant 
                 Kanamycin 
                 Rooting 
                 Examined root 
                 PCR +   
                 roots 
                 Transformation 
               
               
                 Name 
                 type 
                 No. 
                 resistant roots 
                 rate 1   
                 No. with PCR 
                 roots 
                 percentage 2   
                 rates 3   
               
               
                   
               
             
          
           
               
                 pART27 
                 T-DNA 
                 36 
                 105 
                 292% 
                 25 
                 20 
                 80 
                 234% 
               
               
                 pAPn 
                 P-DNA 
                 149 
                 726 
                 487% 
                 37 
                 32 
                 86 
                 419% 
               
               
                 pAPF2rn 
                 P-DNA 
                 106 
                 984 
                 928% 
                 53 
                 51 
                 96 
                 891% 
               
               
                   
               
               
                   1 The rooting rate is the frequency of independent kanamycin-resistant regenerated hairy roots per total number of explants multiplied by 100. 
               
               
                   2 The PCR positive percentage is the number of PCR positive hairy roots per number of examined plants multiplied by 100. 
               
               
                   3 Transformation rate is PCR positive percentage multiplied by rooting rate. 
               
             
          
         
       
     
       Example 4 
       [0028]    Increasing Transformation Rate of Hairy Roots Using pAPF2rn Vector 
         [0029]    To examine effects of MARs on transformation efficiency, two copies of Rb7-MAR were placed in downstream and upstream of pCaMV35S-CodA-Tocs expression cassette using NotI and SacI restriction sites. This expression cassette was produced by cloning of CodA-Tocs in XhoI, NotIalongside of CaMV35S promoter in SacI-XhoI restriction sites of pAPn ( FIGS. 5 and 6 ). 
         [0030]    Sugar beet hairy roots transformations via  Agrobacterium rhizogenes  15834 containing either pAPF2m, pAPn or pART27 were done based on a procedure described EXAMPLE 3. As shown in Table 3, the transformation efficiency of pAPF2rn was 891% and significantly (P&lt;0.05) greater than those of pAPn and pART27 rates for 1.7 and 3.8 fold, respectively. 
         [0031]    While the foregoing has described what are considered to be the best mode and/or other examples, it is understood that various modifications may be made therein and that the subject matter disclosed herein may be implemented in various forms and examples, and that the teachings may be applied in numerous applications, only some of which have been described herein. It is intended by the following claims to claim any and all applications, modifications and variations that fall within the true scope of the present teachings. 
         [0032]    Unless otherwise stated, all measurements, values, ratings, positions, magnitudes, sizes, and other specifications that are set forth in this specification, including in the claims that follow, are approximate, not exact. They are intended to have a reasonable range that is consistent with the functions to which they relate and with what is customary in the art to which they pertain. 
         [0033]    The scope of protection is limited solely by the claims that now follow. That scope is intended and should be interpreted to be as broad as is consistent with the ordinary meaning of the language that is used in the claims when interpreted in light of this specification and the prosecution history that follows and to encompass all structural and functional equivalents. Notwithstanding, none of the claims are intended to embrace subject matter that fails to satisfy the requirement of Sections 101, 102, or 103 of the Patent Act, nor should they be interpreted in such a way. Any unintended embracement of such subject matter is hereby disclaimed. 
         [0034]    Except as stated immediately above, nothing that has been stated or illustrated is intended or should be interpreted to cause a dedication of any component, step, feature, object, benefit, advantage, or equivalent to the public, regardless of whether it is or is not recited in the claims. 
         [0035]    It will be understood that the terms and expressions used herein have the ordinary meaning as is accorded to such terms and expressions with respect to their corresponding respective areas of inquiry and study except where specific meanings have otherwise been set forth herein. Relational terms such as first and second and the like may be used solely to distinguish one entity or action from another without necessarily requiring or implying any actual such relationship or order between such entities or actions. The terms “comprises,” “comprising,” or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. An element proceeded by “a” or “an” does not, without further constraints, preclude the existence of additional identical elements in the process, method, article, or apparatus that comprises the element. 
         [0036]    The Abstract of the Disclosure is provided to allow the reader to quickly ascertain the nature of the technical disclosure. It is submitted with the understanding that it will not be used to interpret or limit the scope or meaning of the claims. In addition, in the foregoing Detailed Description, it can be seen that various features are grouped together in various implementations. This is for purposes of streamlining the disclosure, and is not to be interpreted as reflecting an intention that the claimed implementations require more features than are expressly recited in each claim. Rather, as the following claims reflect, inventive subject matter lies in less than all features of a single disclosed implementation. Thus, the following claims are hereby incorporated into the Detailed Description, with each claim standing on its own as a separately claimed subject matter.