Isolation and characterization of an isoamylase gene from rye

Ke Zheng,Jie Xu,Qingto Jing,André Lroche,Yuming Wei,Youling Zheng,*,Zhenxing Lu,*

1.Introduction

Rye (Secale cereale L.) is an important cereal crop worldwide.Rye grain is mainly used for animal feed,to make beer,whisky or vodka,and is also milled into flour for bread,pumpernickel or crisp bread [1].Compared to other cereal crops such as wheat (Triticum aestivum L.),barley (Horderum vulgare L.) and oat (Avena sativa L.),rye has a number of positive and special attributes,such as outstanding cold hardiness,excellent drought tolerance and strong disease resistance.Apart from its use as a minor cereal crop and a donor of the R genome to triticale(×Triticosecale),it has also been extensively used as an important germplasm source to introgress resistance genes into wheat [2].Some rye attributes are conserved in triticale,an artificial hybrid species made by crossing wheat and rye[3].Triticale is being explored for use as a novel bioindustrial crop in Canada.

Starch synthesis is a complicated process in plants.The first step takes place inside and/or outside amylopasts via ADP-glucose pyrophosphorylase (AGPase,EC 2.7.7.27) for synthesis of ADP glucose,an activated glucosyl donor for starch synthesis[4–6].Subsequent steps lead to two separate pathways for amylose or amylopectin synthesis.Granule-bound starch synthase (GBSS,EC 2.4.1.21),also known as waxy protein,is responsible for the synthesis of amylose polymers [6–8].Amylopectin synthesis results from the elongation of glucan chains with both α-(1,4)-linkage and α-(1,6)-linkage synthesized by the multiple subunits or isoforms of starch synthase(SS,EC 2.4.1.21),starch-branching enzyme (SBE,EC 2.4.1.18) [9,10] and starch debranching enzymes(DBE).According to their different substrate specificities,DBEs are divided into two types:isoamylase (EC 3.2.1.68) and pullulanase (EC 3.2.1.41) [9,11].Genotypic mutants with low starch but high water-soluble polysaccharides were identified in maize(Zea mays L.)[5,12],rice(Oryza sativa L.) [13],barley [14] and Arabidopsis thaliana [15,16],demonstrating that DBEs,in conjunction with SS and SBE,play an essential role in development and accumulation of amylopectin [8,17].Characterization of barley mutants,transgenic potato and rice also indicate that isoamylase plays a crucial role in initiating the development of starch granules[14,18,19].

Starch is the most important carbohydrate in crop grains,but gene interaction in starch synthesis and accumulation in polyploid crops has not been well explored.Since rye has contributed one third of the hexaploid triticale genome,rye isoamylase must be one of the essential enzymes for amylopectin synthesis in triticale grains.However,there is no scientific report about the molecular features of rye isoamylase genes available in public databases.In this study,we isolated genomic and cDNA sequences of a rye isoamylase gene,characterized its structure,domains and expression profiles,and its predicted protein,and also analyzed the evolutionary relationship of isoamylase proteins from rye and other plant species.This is the first report on identification and characterization of an isoamylase gene from the rye genome.

2.Materials and methods

2.1.Plant materials

Hexaploid spring wheat (Triticum aestivum L.) cv.Chinese Spring and diploid spring rye (Secale cereale L.) cv.Rogo were grown under controlled environmental conditions(24 °C day,20 °C night with a 16 h photoperiod of 240 μmol m-2s-1) in the same growth cabinet.Various plant materials (stem,leaf,root,seed) were sampled,flash frozen in liquid nitrogen,and stored at-80 °C until used.

2.2.Preparation of genomic DNA and RNA

Genomic DNA was extracted from young leaf tissue at Zadoks growth Stage 22 [20] using a DNeasy Plant Mini Kit (Cat.No.69104,Qiagen Inc.,Mississauga,ON,Canada).Total RNA was isolated from immature seeds (12 days post anthesis,DPA)according to a phenol/SDS protocol [21].RNA was further purified using the RNeasy Plant Min Kit (Cat.No.74904,Qiagen Inc.,Mississauga,ON,Canada).

2.3.Primers and PCR amplification

Primers for cloning the rye isoamylase gene were designed according to the conserved regions of Aegilops tauschii isoamylase gene sequence (GenBank accession no.AF548379)[22],wheat iso1 mRNA sequence (GenBank accession no.AJ301647)[23]and barley isoamylase mRNA sequence(GenBank accession no.AF490375) [14].Ten pairs of primers were designed to amplify the overlapping genomic DNA sequences that correspond to the rye isoamylase gene.Furthermore,three pairs of primers were developed to amplify the overlapping cDNA sequences.Typically,25 μL of PCR mixture contained 20 pmol primers,30 ng of genomic DNA or 5 μg of cDNA,1 × buffer,1 × Q-solution and 1.25 U of Qiagen HotStar HiFidelity Polymerase (Cat.No.202605,Qiagen Inc.,Mississauga,ON,Canada).Reverse transcription(RT)-PCR was performed using total RNA as the template with Superscript III Reverse Transcriptase (Cat.No.18080-093,Invitrogen,Burlington,ON,Canada).Primer sequences and PCR conditions are listed in Table 1.

2.4.Cloning and sequence analysis

Amplified isoamylase DNA fragments were cloned into the PCR4-TOPO vector (Cat.No.K4575-02,Invitrogen,Burlington,ON,Canada) and at least three independent clones for each fragment were sequenced in both directions by the DNA Sequencing Service Centre,University of Calgary (Calgary,Canada).Rye isoamylase sequences and the corresponding protein were blasted with the NCBI BLASTN tool (http://blast.ncbi.nlm.nih.gov) and aligned with previously reported isoamylase sequences using DNAMAN software v5.0 (Lynnon Biosoft,U.S.A.).The putative encoding regions of transit peptides and mature proteins of isoamylase genes from different plant genomes were predicted using the ChloroP 1.1 server(http://www.cbs.dtu.dk/services/ChloroP/).

2.5.qRT-PCR and gene expression

Total RNAs were isolated from rye leaves,stems,roots and rye seeds at different developmental stages (9,15,24 and 33 DPA)with an RNA Extraction Kit (Cat No.74904,Qiagen Inc.,Mississauga,ON,Canada).For RT-PCR,1 μg of total RNA was transcribed to cDNA in a 20 μL PCR by using the oligo (dT) 18 primer and the SuperScript III Reverse Transcriptase (Cat No.18080-044,Invitrogen,Burlington,ON,Canada).The cDNA mixture was diluted 20 times in RNA-free water and 2.5 μL of cDNA was used as template in a 10 μL real-time PCR consisting of 1 μL primer pair(final concentrate 500 nmol L-1each),1.5 μL RNA-free water and 5 μL SYBR Green (Cat.No.204145,Qiagen,Mississauga,ON,Canada).The primer pair (5′-AAGGAGTGCG AGGGTCTTGG-3′ and 5′-GGTAAGTGGCTGGTGTTGAAGG-3′)was designed by using the Beacon Designer software (v7.0) to detect the transcription level of the rye isoamylase gene identified in this study (GenBank accession no.FJ491379).The real-time PCR was performed on a 7900H qRT-PCR system(Applied Biosystem Canada,Ontario,Canada)and temperature cycling parameters were as follows: 95 °C for 15 min,then 40 cycles of 95 °C for 30 s,60 °C for 30 s and 72 °C for 30 s.Relative expression of rye isoamylase was analyzed by REST software [30].The ADP-ribosylation factor (Ta 2291,GenBank accession no.AB050957),shown to exhibit a constant transcript level at different plant developmental stages under various environmental conductions[31],was used as a reference gene to normalize the rye isoamylase expression.Expression values were subjected to a random variance Mel t-test (P ＜0.05) and twofold or greater differences were considered as differentially expressed.

Table 1-Primer sequences and PCR conditions for the rye isoamylase gene with amplification parameters and product amplicon sizes.

2.6.Construction of a phylogenetic tree

A phylogenetic tree of isoamylase genes was obtained by analyzing the deduced amino acid(aa)sequence from rye(this study),wheat[23],Ae.tauschii[22],barley[14],maize[5],rice[24],pea (GenBank accession no.DQ092413-415),potato [25],sweet potato[26]and Arabidopsis(GenBank accession no.AF002109).A dendrogram was constructed using the Neighbor-Joining method in MEGA 4.1 software and[27].

3.Results and Discussion

3.1.Isolation of a rye isoamylase gene

We amplified PCR and RT-PCR fragments from rye genomic DNA and cDNA using 13 PCR primer pairs designed from conserved domain sequences of plant isoamylase genes(Fig.1).More than one band was amplified with primers Rye-ISA-F14/R14,Rye-ISA-F31/R31,Rye-ISA-F12/R12 and Rye-ISA-F21/R21.In each case,the largest fragment corresponding to the theoretical fragment size (Table 1) was selected for further cloning and sequencing.Ten different overlapping genomic DNA fragments covering the whole rye isoamylase gene and three overlapping cDNA fragments for full-length contigs were isolated and independently cloned into the TOPO-vector and sequenced.Whole genomic DNA and cDNA sequences of the rye isoamylase gene were obtained by the assembly of these overlapping PCR fragments.We determined that the full-length of the rye isoamylase gene was 7351 bp for genomic DNA (GenBank accession no.FJ491378) and 2364 bp for cDNA (GenBank accession no.FJ491379).The overlapping sequences of the genomic DNA and cDNA were identical(data not shown).

Our results demonstrated that the rye isoamylase gene was isolated and cloned with the help of sequence homologies of the same genes from the Ae.tauschii,wheat,and barley genomes,as cereal crops share similar genotypes with highly conserved gene sequences.We found that PCR amplification of the isoamylase gene from the wheat genome was relatively less productive,with no or weak amplicons in comparison with rye(Fig.1).Plausible explanations for such low efficiency may be due to the large hexaploid wheat genome,that is triple the size of rye;PCR efficiency in wheat might be limited by interference of multiple gene loci or by relatively less DNA templates provided by the target genes.Further improvements on PCR conditions and primer designs will be necessary if new isoamylase genes are to be isolated from the wheat genome.

Fig.1-DNA banding patterns of wheat and rye isoamylase genes amplified by different PCR primer sets.Lanes 1 and 2:Rye-ISA-F1/R1; Lanes 3 and 4: Rye-ISA-F14/R14; Lanes 5 and 6: Rye-ISA-F3/R3; Lanes 7 and 8: Rye-ISA-F4/R4; Lanes 9 and 10: Rye-ISA-F25/R25;Lanes 11 and 12:Rye-ISA-F31/R31;Lanes 13 and 14:Rye-ISA-F29/R29;Lanes 15 and 16:Rye-ISA-F30/R30;Lanes 17 and 18:Rye-ISA-F12/R12;Lanes 19 and 20:Rye-ISA-F13R13;Lane 21:Rye-ISA-F1/R20;Lane 22:Rye-ISA-F21/R21;and Lane 23:Rye-ISA-F13/R13.The templates of Chinese Spring genomic DNA were used to amplify the products in lanes 1,3,5,7,9,11,13,15,17 and 19;whereas Rogo genomic DNA templates were used to amplify the products in lanes 2,4,6,8,10,12,14,16,18 and 20 and Rogo cDNA was used in lanes 21,22 and 23.M is the GeneRuler DNA Ladder(Fermentas,Thermo Fisher Scientific).

3.2.Characterization of the rye isoamylase gene

We aligned the genomic and cDNA sequences of the rye isoamylase gene and found that the rye isoamylase gene has 18 exons interrupted by 17 introns.Such intron and exon patterns are nearly identical between the rye and Ae.tauschii genes.The exon lengths of the rye isoamylase gene vary from 72 bp to 363 bp; whereas the intron lengths vary from 73 to 1052 bp.In rice,maize and Arabidopsis,18 exons were identified,but the intron lengths are variable(Fig.2).A comparison of exon sizes among rye,rice,maize,Ae.tauschii and Arabidopsis revealed that these isoamylase genes have identical exon sizes apart from a few differences(Table 2).The first and last exon sizes of the isoamylase genes vary among different plant genomes;exon 2 of the isoamylase gene in rye is 3 bp shorter than that in maize,but exon 16 in rye is 3 bp larger than that in rice and Ae.tauschii.Dinucleotide sequences at the 5′ and 3′ ends in each of the 17 introns were found to follow the universal GT-AG rule[28].

A transit peptide in addition to mature protein regions is normally encoded by plant nuclear isoamylase genes.The cDNA lengths for the transit peptide and the mature protein of rye isoamylase gene are 144 bp and 2220 bp,respectively,and exhibit similarity to other plant isoamylase genes available in public databases.Comparative studies of isoamylase genes among rye and other plant species indicated that mature proteins have higher homology than transit peptides among plant isoamylase genes and the identity of aa sequences between rye,Ae.tauschii,wheat and barley is more than 95%(Table 3).We found that sequence differences in the exon regions of plant isoamylase genes are mainly due to nucleotide substitutions,deletions or insertions.Similarly,differences in the intron regions of plant isoamylase genes are due to more frequent substitution,insertion or deletion events.We determined that DNA homologies range from 40% to 71% in intron regions of isoamylase genes between rye and Ae.tauschii,rice and maize (Table 3),considerably lower than in exon regions.Our results indicated that DNA sequences are highly conserved in the exons of plant isoamylase genes and that evolution rates in the introns of plant isoamylase genes are faster than in the exons.

Fig.2-Schematic diagram for the exon-intron arrangement of isoamylase genes from cereals and Arabidopsis.The sequences used in this analysis were as follows: rye ISA (GenBank accession no.FJ491378),rice OsISA(GenBank accession no.AB093426),Ae.tauschii wDBEI-D1 (GenBank accession no.AF548379),maize SU1 (GenBank accession no.AF030882)and Arabidopsis ISA(GenBank accession no.AF002109).The filled rectangles indicate the exon locations in isoamylase genes.Scale bar,1 kb.

Table 2-Exon lengths (bp) of isoamylase genes from cereals and Arabidopsis.a

Fig.3-Expression profiles of the rye isoamylase gene among(A)various tissues(stem,leaf,root and seed)and(B)different seed development stages (9,15,24 and 33 DPA).Relative expression of rye isoamylase was analyzed by REST software and real-time RT-PCR quantifications were normalized by the ADP-ribosylation factor (Ta 2291,GenBank accession no.AB050957).Expression values were subjected to a random variance Mel t-test(P ＜0.05)and twofold or greater differences were considered as differentially expressed.Error bars represent the SE for three independent replicates.

3.3.Analysis of rye isoamylase proteins

From the full-lengths of genomic DNA and cDNA sequences,we deduced that the rye isoamylase mature protein consists of 787 aa residues with an additional 48 aa for its transit peptide.The calculated molecular weights for the transit peptide and mature protein of rye isoamylase are 5.21 kD and 83.56 kD,respectively.The predicted pI for the mature isoamylase is 5.46.

Table 3-Sequence homologies and similarities of cereal isoamylase genesa (%).

The aa sequences of mature isoamylases exhibited more than 83% homology among rye and other plant genomes,but especially more than 95%homology between rye and Ae.tauschii,wheat and barley.However,sequence homologies for the transit peptides of isoamylases between rye and rice or maize are 31.75% or 27.59%,respectively,significantly less than similar comparisons for the mature proteins (83.31% or 87.18%,respectively) (Table 3).Our results indicate that the structural conservation of the transit peptides for this enzyme is generally lower than that of the mature proteins.Since the transit peptides are the N-terminal aa presequences that direct proteins to an organelle (e.g.,chloroplast,mitochondria) and are required for their transport across membranes from their synthesis sites in the cytoplasm [29],significant diversities in transit peptides of isoamylase between rye and rice or maize may be related to their different cellular structures and metabolic functions,although the mature isoamylases share similar catalytic domains and elements.

3.4.Expression of the rye isoamylase gene

We used quantitative real-time PCR to analyze the expression of the rye isoamylase gene in various tissues and at different seed developmental stages.Our results showed that the isoamylase gene is expressed in all rye tissues tested in this study,with seeds having significantly higher levels of isoamylase transcript than leaves,stems and roots(Fig.3-A).A recent study showed that the ISA1 transcript level is relatively abundant in maize tissues where starch is synthesized [32].As the leaf and other green tissues are temporary storage places for starch accumulation during photosynthesis,the expression of the isoamylase gene in rye leaves and stems demonstrated that amylase may have an important role for either starch synthesis or starch degradation.Isoamylase is termed as the debranching enzyme,essential for formation of crystalline amylopectin [6].We analyzed the expression profiles of the rye isoamylase gene during endosperm development and found that its expression in rye endosperm reached a maximum level at the mid-development stage(15 DPA) and then dropped through 24 and 33 DPA (Fig.3-B).Consistent with previous reports on wheat and maize[23,32],our results confirmed that the isoforms of isoamylase-type DBE genes are maximally expressed during endosperm development and then gradually decline during grain maturation.Studies on barley mutants and transgenic rice suggested that isoamylases play a crucial role in synthesis of phytoglycogen and starch granule structure and initiation [14,19].It will be informative to further investigate the function of isoamylase in granule initiation and degradation in rye and triticale.

To explore evolutionary relationships we constructed a phylogenetic tree on the basis of the aa sequences of mature plant isoamylases.All monocots gathered in a single cluster(Fig.4).There is 98% sequence homology between Ae.tauschii wDBE1 and wheat iso1.On the phylogenetic tree of the deduced mature protein sequences,rye ISA shares 96% sequence homologies with Ae.tauschii wDBE1 and wheat iso1,and 92%homology with barley ISA1,indicating that rye isoamylase is more closely related to Ae.tauschii wDBE1 and wheat iso1.

Fig.4-Phylogenetic tree for isoamylase mature proteins from monocot and dicot plants.The aa sequence of rye isoamylase protein was determined in this study and other plant sequences were obtained from the NCBI GenBank databases.The sequences used for analysis were:rye ISA(FJ491378/FJ491379),Ae.tauschii wDBE1(AF548379),wheat iso1(AJ301647),barley ISA1(AF490375),rice OsISA(AB093426),maize SU1(AF030882),Arabidopsis ISA(AF002109),sweet potato iso(DQ074643),potato iso1(AY132996),potato iso2(AY132997),potato iso3(AY132998),pea iso1(DQ092413),pea iso2(DQ092414)and pea iso3(DQ092415).All sequences were analyzed by the Neighbor-Joining method in MEGA 4.1 software[27].

4.Conclusions

In this study,we isolated and characterized genomic DNA and cDNA and also predicted the corresponding protein sequence of the rye isoamylase gene.By comparing isoamylase genes and their proteins among rye and other plant species,we found that plant isoamylase genes are highly homologous in the exon regions and rye isoamylase is most closely homologous in aa sequence to wheat and Ae.tauschii than to barley in terms of phylogenetic relationship.Our real-time PCR results indicated that the rye isoamylase gene is mainly expressed in seed endosperms with a maximum level at the mid-development stage (15 DPA).Starch synthesis is a complicated metabolic system in plants and characterization of starch synthesis genes is essential for establishing a basis to explore starch structure,function,and accumulation.Isoamylase genes have been isolated and characterized from different plant species,but their precise roles in starch synthesis and granule initiation are not yet clear.The rye isoamylase isolated and characterized in this study has provided new and essential information to explore its function in amylopectin accumulation in rye and triticale grains and also its effects on subsequent development of new triticale genotypes for novel starch granule types leading to higher or lower amylopectin contents.

This study was supported by the MOE-AAFC PhD Research Program and partial A-Base funding from Agriculture and Agri-Food Canada.

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