Isolation, cloning, and characterization of a partial novel aro A gene in common reed (Phragmites australis)

Figures & data

Figure 1. Phragmites australis partial aro A gene amplification. The PCR amplification of partial aro A gene is shown in lanes 2 and 3. A 1 Kb MW marker is shown in lane 1.

Figure 2. Analysis of the pTZ carrying the aro A gene. The PCR-amplified insert of the plasmid is shown in lanes 3–5 and plasmid without insert (negative control) is shown in lane 2. A 1 kb MW marker is shown in lane 1. Plasmid of lane 3 was selected to sequence.

Figure 3. The partial sequence of Phragmites australis with intron regions. The underlined regions are primers (forward (TKZF) and reverse (TKZR)). The regions in gray box are introns (86 bp and 289 bp).

Figure 4. The partial sequence of aro A of Phragmites australis without intron regions. The underline regions are primers (forward (TKZF) and reverse (TKZR)).

Figure 5. The phylogenic relationship of Phragmites australis with other species. A phylogenic tree (unrooted) based on the genetic distance of amino acid sequences was constructed by the Clustal method with using the DNAstar software.

Table 1. Selected sequences from NCBI for multiple sequence alignment analysis.

Accession number	Scientific name
HQ403648.1	Eleusine indica (epsps-R)
HQ403647.1	Eleusine indica (epsps-S)
X63374.1	Zea mays
GU256771.1	Zoysia japonica
XM_002436379.1	Sorghom bicolor
EU977181.1	Triticum aestivum
AJ310166.3	Lolium rigidum
AF413081.1	Oriza sativa
JN160845.1	Capsicum annuum cultivar Fogo

Table 2. Sequence comparison of the conserved region of several aro A in organisms and Phragmites australis. Besides P. australis Smith, others have previously reported by Gasser et al. (1987), Klee et al. (1987), and Padgette et al. (1991).

EPSPS sources	Sequence
Escherchia coli	L^90 FLGNAG^96TAMRP^101L^102
Salmonella typhimuruim	L^90 FLGNAG^96TAMRP^101L^102
Petunia hybrid	L^95 FLGNAG^101TAMRP^106L^107
Lycopersicon esculentum	L^95 FLGNAG^101TAMRP^106L^107
Arabidopsis thaliana	L^95 FLGNAG^101TAMRP^106L^107
Brassica napus	L^95 FLGNAG^101TAMRP^106L^107
Zea mays	L^95 FLGNAG^101TAMRP^106L^107
Aspergillus nidulans	L YLGNAG TASRF L
Saccharomyces cerevisiae	L YLGNAG TASRF L
Phragmites australis	FLGNAG TAMRP L
Consensus sequence	L XLGNAG TAXRX L

Table 3. Amino acid composition of partial EPSPS gene of Phragmites australis.

Amino acid	Number	Percent
Ala (A)	12	12.2
Arg (R)	5	5.1
Asn (N)	2	2.0
Asp (D)	4	4.1
Cys (C)	2	2.0
Gln (Q)	2	2.0
Glu (E)	2	2.0
Gly (G)	15	15.3
His (H)	0	0.0
Ile (I)	3	3.1
Leu (L)	14	14.3
Lys (K)	4	4.1
Met (M)	3	3.1
Phe (F)	2	2.0
Pro (P)	7	7.1
Ser (S)	5	5.1
Thr (T)	5	5.1
Trp (W)	0	0.0
Tyr (Y)	2	2.0
Val (V)	9	9.2
Pyl (O)	0	0.0
Sec (U)	0	0.0

Bold suggests that Glycine (Gly) with 15.3% has important role into protein biosynthesis such as EPSPs catalytic reaction.

Gasser SC, Winter JA, Hironaka CM, Shah DM. (1987). Structure, expression and evolution of the 5-enolpyruvylshikimate-3-phosphate synthase genes of petunia and tomato. J Biol Chem 263:4280–9

 Web of Science ®Google Scholar

Klee HJ, Muskopf YM, Gasser CS. (1987). Cloning of an Arabidopsis thaliana gene encoding 5-enolpyruvylshikimate-3-phosphate synthase: Sequence analysis and manipulation to obtain glyphosate-tolerant plants. Mol Gen Genet 210:437–42

 PubMedGoogle Scholar

Padgette SR, Biest DR, Gasser CS, et al. (1991). Site-directed mutagenesis of a conserved region of the 5-enolpyruvylshikimate-3-phosphate synthase active site. J Biol Chem 266:22364–9

 PubMed Web of Science ®Google Scholar