Abstract: In order to knowing the variation of starch synthase I (SSI) in Triticum L. and Aegilops and exploiting new gene resources for the improving of starch quality in common wheat, using PCR cloning, eight sequences of SSI genes were obtained from AS60 (DD, Aegilops tauschii), AS2255 (AABB, Triticum turgidum), SHW-L1 (AABBDD, synthetic hexaploid wheat) and Chuanmai 32 (AABBDD, Triticum aestivum), respectively. These sequences of SSI gene showed the higher conservative characterization between different materials. A total of 10 variable nucleotide bases and two substitutions of amino acid residues (Ser/Asn and Ala/Val) were observed in the cloning sequences. Alignment of all sequences, including SSI gene from T. aestivum, Hordeum vulgare, Brachypodium distachyon, Zea mays and Oryza sativa suggested that the sequences from Ae, tauschii, T. turgidum and synthetic hexaploid wheat, were more similar to SSI gene from common wheat and barley and far distance with sequences from Brachypodium distachyon, Zea mays and Oryza sativa. Using neighbor-joining method, phylogenetic tree including a total of 28 sequences could be clustered into four groups. Most of sequences of SSI from different species were clustered together and SSI gene showed the clear difference with other starch synthases, including Granule-bound Starch Synthase (GBSS), SSII, SSIII and SSIV. These results suggested that there was variation fro SSI gene in Triticum L. and Aegilops. It necessary to developing molecular markers for exploit genetic resources and the improving of wheat starch quality.
INTRODUCTION
Starch synthase (SS) plays an important role in the series of synthesis of starch of wheat. SS extend α-1, 4 glucan chains by catalyzing the transfer of the glucosyl moiety of ADP-Glc to the reducing end of a pre-existing α-1,4 glucan primer to synthesize the insoluble glucan polymers amylose and amylopectin. Four soluble starch synthase gene: SSI (Baba et al., 1993; Knight et al., 1998), SSII (Dry et al., 1992; Edwards et al., 1995; Harn et al., 1998), SSIII (Abel et al., 1996; Marshall et al., 1996; Gao et al., 1998) and SSIV are each thought to be primarily involved in amylopectin synthesis and also play the roles in amylose biosynthesis. Each type of starch synthase genes determines different respects in amylopectin synthesis through analysis of the SS mutants. In the mutants of rice and Arabidopsis, SSI showed a distinct capacity for the synthesis of chains with DP of 8-12 from DP of 6 to 7 (Fujita et al., 2006; Delvalle et al., 2005). The existence of functional interactions between the starch branching enzymes (SBEII) and the SSI and SSII were reported in classes of amylopectin synthesis in amyloplasts based on the developing wheat endosperm (Tetlow et al., 2008). Alternatively, loss of SSII specifically also leads to the loss of SSI protein in the granule-bound phase and the effect of this mutation is clearly manifest from the mid-stage of endosperm development in wheat (Kosar-Hashemi et al., 2007).
The isoenzyme of SSI was not reported in the other researches. SSI expressed in the early and middle stage of the development of endosperm (Li et al., 1999). There was conserved sequence in the transit peptide of SSI gene: Lys-Ser-Gly-Gly, which is the site for attached by ADP-Glc (Baba et al., 1993; Tanaka et al., 1995). Soluble starch synthase determined to the chains with degree of polymerization (DP) of 6-15 by the expressing of SS gene and Starch Branching Enzyme (SBE) gene in E. coli (Guan and Keeling, 1998). In rice, SSI and SSIIIa were the main soluble starch synthase and SSI had higher activity than SSIIIa, take 70% activity of soluble synthase (Fujita et al., 2006). The similar performances were reported in maize and wheat (Cao et al., 1999; Li et al., 2000). Gene SSI, encoded protein Spg-3 with 75 KDa of molecular weight, was located on 7AS, 7BS and 7DS in wheat (Yamamori and Endo, 1996).
In the present study, in order to understanding the variation o of SSI gene and exploiting new genetic resources in Triticum L. and Aegilops, the partial sequences of SSI were investigated from Aeilops tauschii, Triticum turgidum, Triticum aestivum and synthetic hexaploid wheat. Phylogenetic analysis was also done for evaluating the relationship between different species and starch synthase genes.
MATERIALS AND METHODS
Materials: A total of four accessions, including AS60, AS2255, SHW-L1 and Chuanmai 32, were used in this study. All the materials were provided by the Germplasm Laboratory of Triticeae Research Institute, Sichuan Agriculture University. Synthetic hexaploid wheat SHW-L1 (AABBDD, T. aestivum) was obtained through distant hybridization between accession AS2255 (AABB, T. turgidum) and AS60 (DD, Ae. tauschii) (Zhang et al., 2004). Variety Chuanmai 32 (AABBDD, T. aestivum) was a commercial common wheat cultivar at southwest of China.
Methods: All materials were planted in the field of Sichuan Agricultural University, China. Total RNA was extracted from the new collected seed after pollination 15 days using the total RNA isolation kit (Takara) according to the manufacturer’s instruction. To eliminate gnomic DNA, extracted RNA was treated with RNAase-free DNase supplement supplied with the kit. RNA quality and purity were tested by UV absorption spectrophotometer and gel electrophoresis. Total RNA was used to synthesize cDNA using the Reverse Transcriptase M-MLV (Takara) and oligo-dT primer according to the manufacturer’s instruction. A pair of primers, which were designed based on the sequence AJ292521 (GeneBank), was used as the cloning primers: the forward primer: 5’-TTATTGGAAGACTGGATTACC-3’ and the reverse primer: 5’-CACAGCCGTCATCTACTCT -3’. The amplified region includes from the 11th exon to the 15th exon and partial 3’UTR. PCR was performed in a 50 μL volume, containing 1.5 UTaq plus DNA polymerase, 100 ng of each cDNA, 5 μL PCR buffer (supplied with Taq plus DNApolymerase), 1.5 mM MgCl2, 100 mM of each dNTP and 150 ng each primer. The reactions were conducted in a PTC-220 (MJ Research, USA) using the following program: 95°C for 3 min, followed by 35 cycles at 94°C for 1 min, at 60°C for 1 min and at 72°C for 2 min and a final extension step of 72°C for 10 min. The PCR products were separated on 1.5% agarose gels. The expected fragments were recovered and cloned into pMD18-T vector (TaKaRa), then transformed into the competent E. coli cells (DH5α). Therefore, positive clones of each accession were sequenced by commercial company (Invitrogen) in two directions.
Data analysis: The obtained sequences were compared with known SSI sequences using BLAST (http://www.ncbi.nlm.nih.gov/BLAST). Sequences were manually inspected with BioEdit ver 7.0.9 (Hall, 1999). Sequence alignment was completed with ClustalX (Larkin et al., 2007) and DNAMAN 5.2.2 (http://www.lynnon.com). Neighbour-joining trees was constructed based on the alignment of all known sequences, including sequences from barley, rice, maize, sorghum and potato respectively, using Kimura 2-parameter model in MEGA4.0 (Sation and Nei, 1987; Tamura et al., 2007).
RESULTS
Variation of sequences of SSI gene: Bands about 750 bp were amplified by PCR from four accessions respectively. After cloning and sequencing these bands, eight sequences were obtained, including one sequence from AS60, two sequences from AS2255, three sequences from Synthetic hexaploid wheat SHW-L1 and two from wheat variety Chuanmai 32. Except from AS2255, the sequences of SSI gene showed the complete same from one sample. Blast in NCBI confirmed all the sequences belonged to the partial SSI gene, suggested that these primers could successfully cloning the SSI gene from T. aestivum and Ae. tauschii. Analysis of these sequences showed 13 variable nucleotide sites in all five sequences, suggested that there was higher conservative for SSI gene between diploid, tetraploid and hexaploid in T. aestivum and Ae. tauschii. Interestingly, 10 variable nucleotide sites were observed in sequence AS2255-2 from Synthetic hexaploid wheat SHW-L1, indicated this sequence was clearly distinguishing with other SSI gene.
Only two variable sites, at 47 (Ser/Asn) of sequence AS2255-2 and 141 (Ala/Val) of sequence SHW-L1, were observed between five sequences through alignment of amino acid residue, further suggested that SSI was very high conserve gene in T. aestivum and Ae. tauschii (Fig. 1). Moreover, the variation of amino acid residues was detected between these five sequence and sequences from T. aestivum (CAB99209), Hordeum vulgare (CAX51357), Brachypodium distachyon (XP003561063), Zea mays (AAB99957) and Oryza sativa (AAP56350). The results showed the sequences from Ae. tauschii, T. turgidum and T. aestivum were more similar to SSI from common wheat and barley. Sequences from other species, Brachypodium distachyon, Zea mays and Oryza sativa showed more variable amino acid residue, comparing with sequences from Triticum L. and Aegilops. But there was no variation on two important functional domains, domain II and domain III, between all sequences. These results suggested that although SSI is a very conservative gene, the variation could be observed between different species.
| Fig. 1: | Alignment of amino acids residues of partial SSI gene, AS60 (Ae. tauschii), AS2255-1 and AS2255-2 (T. turgidum), SHW-L1( Synthetic hexaploid wheat), Chuanmai 32 (T. aestivum) CAB99209 (Triticum aestivum), CX51357 (Hordeum vulgare), XP003561063 (Brachypodium distachyon), AAB99957 (Zea mays), AAP56350 (Oryza sativa) |
Phylogenetic analysis: In order to estimate the relationship of SSI gene between different species, the phylogenetic tree was constructed by neighbor-joining method (Fig. 2). A total of 28 sequences, which included not only the cloning sequences of SSI gene in this study, but also the genes reported in other researches from wheat, barely, rice, maize, maize, sorghum, potato, Arabidopsis and Brachypodium distachyon, respectively. Moreover, Granule-bound Starch Synthase (GBSS), SSII, SSIII and SSIV genes were also applied in this phylogenetic analysis. All the sequences could be clustered to four groups. Group I included the new cloning sequences in this study, the sequences from common wheat and the sequence from sorghum, maize and Brachypodium distachyon. Sequences from Triticum L. and Aegilops were clustered closely than other species in this group. Three sequences from barley, rice and Ae. tauschii, respectively, were clustered into group II. Gene GBSSI, GBSSII and the alleles of SSII were clustered into group III. Genes SSI from Arabidopsis and potato were clustered into group IIIV. This group also included SSIV and SSIII genes from T. aestivum, indicated that SSI gene had more similarity with these two genes.
| Fig. 2: | Neighbour-joining tree based on sequences of SSI gene was constructed by using Kimura 2-parameter model in MEGA 4.0, Bootstrap tree was obtained by 1, 500 repeat calculation |
Phylogenetic tress further suggested that although high conservative characterization of sequence, the variation of SSI gene could be detected clearly between different species and between different starch synthase genes. These results implied that it was possible to developing the special molecular markers to apply in the improving of wheat starch quality.
DISCUSSION
Both of Ae. tauschi and synthetic hexaploid wheat are the very important genetic resources for the breeding of common wheat. Ae. tauschii, which usually considered as the donor of D genome in common wheat (McFadden and Sears, 1946; Kihara, 1944) and contains a lot of excellent potential characters (Kimber and Feldman, 1987), as well as the very useful materials to understand the evolution of common wheat (Dudnikov, 1998). Synthetic hexaploid wheat, produced by crossing tetraplioid whet (AABB) with Ae. tauschii (DD), has been used as an intermediary for transferring resistance genes from the wild ancestor to cultivated wheat (Del Blanco et al., 2001). Synthetic hexaploids have been reported as having resistance to disease (Lan et al., 1997), tolerant to abiotic stresses and quality (Del Blanco et al., 2001; Kunert et al., 2007). Starch, which accounts for 65-75% of wheat grain dry weight, is also widely used in food industry including bread, noodles, biscuits, cakes and non-food industry including paper, plastic, adhesive, textile, medical and pharmaceutical industry (Sestili et al., 2010). The further improving the quality of starch need to exploiting continuously new gene resources. Starch Synthase (SS) I plays an important role in the series of synthesis of starch of wheat, not only involved in amylopectin synthesis and also in amylose biosynthesis. SSI preferentially synthesizes chains of DP 7-11 by elongating amylopectin short chains of DP 4-7. In present study, eight partial sequences of SSI gene were cloned from Ae. tauschii, tetraploid wheat, synthetic hexaploid wheat and common wheat. The results suggested that the variation could be detected between different species, although SSI gene had high conservative characterization. In this study, although the partial sequences of SSI gene were cloned, two substitutions of amino acid residues, Ser/Asn and Ala/Val, were observed in tetraploid AS2255-2 and hexaploid SHW-L1. This result also showed that the variation of SSI gene could be detected in Trticum L. and Aegilops, suggested that it was possible to developing new molecular markers to exploit SSI gene resources. In addition, the effect of these variations for the function of SSI gene and starch quality should be investigated in these accessions.
Plants contain up to five isoforms of starch synthase that are categorized, according to conserved sequence relationships. These five isoforms include GBSS, SSI, SSII, SSIII and SSIV. SSI exclusively involved in amylopectin biosynthesis. In the present study, the phylogenetic tree showed that these five genes could be clustered clearly into different groups. Although came from different species, most of SSI gene sequences were clustered into one group, indicated SSI gene had higher conservative between different species, especially in Triticum L. and Aegilops.
ACKNOWLEDGMENT
This study was supported by the National Basic Research Program of China (973) Program and 2011CB100100) and the Key Project of National Natural Science Foundation of China (31230053).