Functional Characterization of 5-Enopyruvylshikimate-3-Phosphate Synthase from Alkaliphilus metalliredigens in Transgenic Arabidopsis

Abstract

Although a large number of AroA enzymes (EPSPS: 5-enopyruvylshikimate-3-phosphate synthase) have been identified, cloned, and tested for glyphosate resistance, only two AroA variants, derived from Agrobacterium tumefaciens strain CP4 and Zea mays, have been utilized to produce the commercial glyphosate-resistant crops. Here, we have used a PCR-based twostep DNA synthesis method to synthesize an aroA gene ($aroA_{A.\;metalliredigens}$) from Alkaliphilus metalliredigens, encoding a new EPSPS. Furthermore, transgenic Arabidopsis with the new $aroA_{A.\;metalliredigens}$ gene was obtained to confirm the potential of the novel aroA gene in developing glyphosate-resistant crops.

Introduction

Glyphosate, a nonselective broad-spectrum herbicide that blocks plant growth by inhibiting 5-enopyruvylshikimate-3-phosphate synthase (EPSPS, also designated as AroA), has been extensively used over the last three decades. However, one disadvantage of glyphosate is that it can also kill crops [13]. Recent advances in genetic engineering have facilitated the introduction of the glyphosate-insensitive form of EPSPS enzyme into plants using transformation techniques. Therefore, obtaining novel and valid glyphosate-insensitive genes is critical to generating glyphosate-tolerant crops.

In the past years, two classes of promising EPSPS have been cloned, identified, and tested for glyphosate resistance [1,5,7,8]. Although many EPSPS enzymes have been extensively characterized during the past three decades, only two AroA variants, derived from Agrobacterium tumefaciens strain CP4 and Zea mays, have been utilized to produce the commercial glyphosate-resistant crops (U.S. Patent 6,040,497).

The alkaliphilic halophile Alkaliphilus metalliredigens has been studied by many researchers [6], and its complete genomic sequence has been available recently on the NCBI website (GenBank: NC_009633.1). Although many EPSPS enzymes have been extensively characterized during the past three decades, to our knowledge, there are no published reports of EPSPS enzymes from Alkaliphilus metalliredigens. Therefore, in this study, we set out to chemically synthesize a new plant-optimized version of the aroA gene from A. metalliredigens by a PCR-based two-step DNA synthesis (PTDS) method [14], according to the aroAA. metalliredigens gene sequence (GenBank: NC_009633). Furthermore, in order to evaluate its potential, we have transformed the new gene into Arabidopsis by using a floral dip method, and demonstrated that transgenic Arabidopsis plants exhibit significant glyphosate resistance when compared with the wild type.

 

Materials and Methods

Bacterial Strains, Chemicals, and Plant Materials

S3P, glyphosate (free acid form), and PEP were purchased from Sigma Chemical Co., Ltd. (St. Louis, MO, USA). All other chemicals were of analytical grade and obtained from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). E. coli strain ER2799 [2] (with the EPSP synthase gene deleted from its genome) was provided by Dr. Thomas C. Evans, Jr. (New England Biolabs, USA). A. tumefaciens GV3101 and A. thaliana (ecotype Columbia L.) plants were sourced from our laboratory. The latter were grown on Murashige and Skoog medium [10] with 1% agar.

Chemical Synthesis of aroAA. metalliredigens

The sequence of aroAA. metalliredigens was artificially synthesized using a PCR-based two-step DNA synthesis strategy (see primers in Fig. S1 and Fig. 1). All the codons in aroAA. metalliredigens were optimized and preferentially designed for plants. The polymerase chain reaction (PCR) was carried out in a total volume of 50 μl containing 200 nM of each outer primer and 20 nM of each inner primer. The reaction conditions were 30 cycles of 94℃ for 30 sec, 54℃ for 30 sec, and 72℃ for 90 sec. The amplified fragment was digested with BamHI and SacI, cloned into TA clone vector Simple pMD-18, and sequenced [15].

Fig. 1.The PTDS strategy for the synthesis of the aroAA. metalliredigens gene.

Sequence Analysis of aroAA. metalliredigens

The sequence analysis was conducted through database search using the BLAST program (NCBI, National Center for Biotechnology Information, http://www.ncbi.nlm.nih.gov). Amino acid sequence analysis was done using DNAMAN ver. 6.0. A phylogenetic tree was constructed using MEGA ver. 2.1 from CLUSTAL W alignments.

In Vitro Glyphosate Resistance Assay

To assay glyphosate resistance of AroAA. metalliredigens, ER2799 transformants (transformed with aroAA. metalliredigens) were grown in LB medium. After induction in LB medium for 12 h, cells were diluted to 5 × 103 cells/μl. About 2 μl of dilutions was spotted on fresh M9 agar plates supplemented with glyphosate at various concentrations (0, 50, 150, and 200 mM) and then incubated at 30℃ for 2 days.

Construction of the Plant Expression Vector and Plant Transformation

Construction of the plant expression vector and plant transformation were performed as described by Xu et al. [15] and Tian et al. [13]. To ensure that the transgene would be targeted to the chloroplast, the fragment encoding the chloroplast transit peptide of A. thaliana (TSP) was added to the constructs [3,9]. Then the final constructs D35S:TSP: aroAA. metalliredigens :Nos were introduced into A. tumefaciens GV3101 by electroporation, and subsequently transformed into A. thaliana (ecotype Columbia) by a previously described floral dip method to generate transgenic plants [16].

Transgenic Plant Selection

The transgenic nature of the plants was confirmed by PCR analysis of genomic DNA using the specific primers P1Z (5’-cgggatccatgctggtcactaacaaagtc-3’) and P1F (5’-gagctcttagtggtgatggtgat-3’). Then reverse transcription (RT)-PCR was used to determine the level of aroAA. metalliredigens transcription. In order to improve the reliability of RT-PCR, the A. thaliana actin gene (GenBank: U41998) served as an internal standard to normalize the amount of cDNA amplified with primers TactZ (5’-gcaccctgttcttcttaccgag-3’) and TactF (5’-agtaaggtcacgtccagcaagg-3’). Specific DNA fragments (~1,300 bp) of the aroAA. metalliredigens gene was then amplified from the transgenic plants using the same amount of cDNA. PCR products were separated on 2% (w/v) agarose gels and quantified using a Model Gel Doc 1000 (Bio-Rad, USA). The expression patterns of the aroAA. metalliredigens gene were evaluated with a Shine Tech Gel Analyzer (Shanghai Shine Science of Technology Co., Ltd., China).

Assay of Glyphosate Resistance

For the assay of germination, the T3-sterilized A. thaliana seeds were grown directly on Murashige and Skoog (MS) medium containing various glyphosate concentrations (0, 200, 500, and 1,000 μM) in Petri dishes under a controlled-environment chamber (22°C, 16:8 h day:night cycle). For the assay of root growth, the T3 sterilized A. thaliana seeds were grown vertically on MS medium containing glyphosate (0, 200, 500, and 1,000 μM) in Petri dishes. After 2 weeks of growth, the photos were taken.

 

Results and Discussion

Chemical Synthesis of aroAA. metalliredigens

The aroAA. metalliredigens was synthesized by the PTDS method. The experimental strategy of PTDS is outlined in Fig. 1. Errors in the synthetic gene were corrected by the overlap extension polymerase chain reaction (OE-PCR) method [11]. Forty-four oligonucleotides were used to synthesize the recombinant gene (Fig. S1) based on the aroAA. metalliredigens gene sequence (GenBank: NC_009633). BLAST search showed that the synthesized recombinant aroAA. metalliredigens gene was 100% identical to the wild-type aroAA. metalliredigens gene.

Sequence Analysis of aroAA. metalliredigens

Amino acid sequence analysis indicated that AroAA. metalliredigens shared more than 41.87% amino acid identity with AroAA. tumefaciens CP4 (GenBank: Q9R4E4), but less than 22.89% amino acid identity with AroAE. coli (GenBank: P07638). Moreover, the residues involving PEP and S3P binding domains in AroAA. metalliredigensand AroAA. tumefaciens CP4 are all highly conserved. Phylogenetic analysis indicated that AroAA. metalliredigens was a Class II EPSPS (Fig. 2).

Fig. 2.Sequence analysis of AroAA. metalliredigens. (A) Amino acid sequence alignment of AroAA. metalliredigens, AroA E. coli, and AroAA. tumefaciens CP4. Asterisks and circles: residues important for S3P binding and PEP binding in AroAE.coli, respectively. Important domains for glyphosate tolerance and maintenance of productive PEP binding in class II AroA are boxed. (B) Phylogenetic tree analysis of AroAA. metalliredigens using MEGA. Class I AroA proteins shown in this Fig are from E. coli (P07638), Aeromonas salmonicida (Q03321), Arabidopsis thaliana (P05466), Nicotiana tabacum (P23981), Petunia hybrida (P11043), Zea mays (CAA44974), and Bordetella pertussis (P12421); Class II AroA proteins shown in this Fig are from Pseudomonas sp. strain PG2982 (P56952), Agrobacterium sp. CP4 (Q9R4E4), Staphylococcus aureus (Q05615), Dichelobacter nodosus (Q46550), Ochrobactrum anthropi (GenBank:GU992200), Bacillus cereus (GenBank: HQ419071), and Streptococcus pneumoniae (Q9S400).

In Vitro Glyphosate Resistance Assay

To furthur identify the ability of AroAA. metalliredigens, in vitro glyphosate resistance assay was assessed by a drop test on M9 solid medium supplemented with increasing concentrations of glyphosate. As shown in Fig. 3, we observed that on M9 medium, 150 mM glyphosate was preliminarily to inhibit the growth of AroAA. metalliredigens cells. At 200 mM glyphosate, AroAA. metalliredigens cells still proliferated, suggesting AroAA. metalliredigens has relatively higher tolerance to glyphosate.

Fig. 3.Assay of glyphosate resistance in vitro. Dilutions (2 μl) were spotted on M9 medium with various glyphosate concentrations (0, 50, 150, and 200 mM). Data shown are representative of three independent experiments.

Transgenic Plant Selection

Transgenic A. thaliana was used to evaluate the potential application of AroAA. metalliredigens in developing glyphosate-resistant crops. Eight transgenic plants with aroAA. metalliredigens were obtained, and transgenic plants of the T3 generation were confirmed by PCR analysis of genomic DNA. Then, three transgenic lines (named Am1, Am2, and Am3) were further analyzed for gene expression using RT-PCR analysis as previously described. First, the A. thaliana actin gene (GenBank: U41998) from the three transgenic lines (Am1, Am2, and Am3) and the wild type was amplified to ensure that the same amount of cDNA was used to amplify the target genes in the transgenic lines. Then, the specific DNA fragments (~ 1,300 bp) of aroAA. metalliredigens were amplified from the transgenic lines. Agarose gel electrophoresis showed that the DNA intensity of aroAA. metalliredigens in the three different transgenic lines was the same (Fig. 4), confirming that the level of transcription of aroAA. metalliredigens in the three different transgenic lines was equal. Moreover, RT-PCR demonstrated that the inserted genes were actively and stably transcribed in the transgenic plants.

Fig. 4.Expression level of aroAA. metalliredigens cDNAs in different transgenic lines. Each lane contained 4 μl of RT-PCR products obtained using total RNA extracted from three-week-old plants grown under normal conditions. WT, wild type; Am, transgenic Arabidopsis lines with aroAA. metalliredigens gene. Data shown are representative of three independent experiments.

Assay of Glyphosate Resistance

Glyphosate can affect seed germination and root development. Seeds and roots are usually poorly developed under glyphosate stress [4,12,17]. Thus, the assays on the germination and root growth of transgenic A. thaliana were evaluated in this study. After 2 weeks, the photos were taken, as shown in Fig. 5. It shows that aroAA. metalliredigens -transgenic plants germinated well in 1,000 μM glyphosate, whereas control plants did not germinate at 200 μM. It is obvious that aroAA. metalliredigens -transgenic plants were more tolerant to glyphosate than control plants, which was further confirmed by the assay of root growth. As shown in Fig. 5B, the root growth of aroAA. metalliredigens -transgenic plants could grow at 1,000 μM glyphosate, whereas control plants were strongly inhibited at 200 μM glyphosate.

Fig. 5.(A) The comparative germination image of transgenic seeds on MS medium containing various glyphosate concentrations (0, 200, 500, 1,000 μM) in Petri dishes. (B) The comparative image of root length between different transgenic lines on MS medium containing various glyphosate (0, 200, 500, 1,000 μM) in Petri dish.

In conclusion, the results indicate that the Arabidopsis transformation with the new aroA gene is more resistant to glyphosate exposure than control plants. Thus, the novel gene can be applied to transgenic crops with glyphosate tolerance in the future.