Structure and expression analysis of genes encoding ADP-glucose pyrophosphorylase large subunit in wheat and its relatives
ADP-glucose pyrophosphorylase (AGP), which consists of two large subunits (AGP-L) and two small subunits (AGP-S), controls the rate-limiting step in the starch biosynthetic pathway. In this study, a full-length open reading frame (ORF) of AGP-L gene (named as Agp2) in wheat and a series of Agp2 gene sequences in wheat relatives were isolated. The coding region of Agp2 contained 15 exons and 14 introns including a full-length ORF of 1566 nucleotides, and the deduced protein contained 522 amino
... ntained 522 amino acids (57.8 kDa). Generally, the phylogenetic tree of Agp2 indicated that sequences from A and D genome donor species were most similar to each other and sequences from B genome donor species contained more variation. Starch accumulation and Agp2 expression in wheat grains reached their peak at 21 and 15 days post anthesis (DPA), respectively. Page 3 of 22 https://mc06.manuscriptcentral.com/genome-pubs Genome genomes, so they could contribute to research on functional genes. To date, no Agp2 sequences have been reported from relatives of common wheat. Materials and methods Plant material Triticum aestivum cv. 'Bobwhite 7' (AABBDD, 2n = 42) and 18 accessions of Triticum urartu (A u A u genome, 2n = 14), Triticum monococcum L. (A m A m , 2n = 14), Aegilops speltoides (SS, 2n = 14), Aegilops bicornis (Forssk.) Jaub. & Spach (S b S b , 2n = 14), Aegilops longissima Schweinf. & Muschl. (S l S l , 2n = 14), Aegilops sharonensis Eig (S sh S sh , 2n = 14), and Aegilops tauschii (DD, 2n = 14) were kindly provided by USDA-ARS (http://www.arsgrin.gov) ( Table 1 ). The accessions were grown in a glasshouse with a daytime temperature of 23°C and a nighttime temperature of 18°C, with a photoperiod of 16 h. Wheat seeds were collected at different developmental stages and stored at −80°C until DNA and RNA were extracted. Three replicates were used for all samples. Isolation of the Agp2 open reading frame (ORF) and gene sequences Genomic DNA and total RNA were extracted from endosperm at 20 days post anthesis (DPA), using the modified SDS method described by Aljanabi and Martinez (1997) and Trizol (Tiangen, Beijing, China), respectively. cDNA was obtained from reverse transcription using a PrimeScript™ 1st Strand cDNA Synthesis Kit (Takara, Dalian, China). A set of primers (Table 2) was designed from the cDNA sequence of Agp2 in wheat (Ainsworth et al. 1995) to isolate the ORF or gene sequences of Agp2 from wheat and 18 diploid Triticeae accessions. PCRs were performed in 50-µL reactions containing 5 U PrimerStar HS DNA polymerase, 25 µL 2× PrimeSTAR GC Buffer (Mg 2+ Plus), 200 µM of each dNTP, 200 nM of each primer, and 100 ng of DNA. The PCR program included an initial denaturation at 94°C for 5 min, followed by 35 cycles of 94°C for 1 min, 60°C for 1 min, 72°C for 2 min, and a final extension at 72°C for 10 min. Target bands were purified and cloned into the pEasy-T1 (Transgen, Beijing, China) vector and then transformed into E. coli (DH5α) (Transgen, Beijing, China). For each target band, three positive clones were selected and sequenced by Invitrogen (Shanghai, China).