Study on cloning of Starch Synthase III gene in wheat
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Date
2019
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Dr. Rajendra Prasad Central Agricultural University, Pusa, Samastipur
Abstract
Bread wheat is one of the primary sources of energy and proteins for millions of people world over. Starch is the most critical food energy source in the world and constitutes 65% to 80% proportion of the wheat grain weight. Starch synthase III is directly associated with starch accumulation in wheat. Hence, the present investigation entitled “Study on cloning of Starch Synthase III gene in wheat” was conducted to clone the full length of starch synthase III genes. The work includes detailed In-silico characterization of genome-specific TaSS-III genes in wheat, covering positions of exons, introns, location of the genes in chromosomes, phylogenetic analysis, protein’s domain analysis, and expression analysis under heat stress conditions. The genomic DNA of heat susceptible PBW-343 and tolerant KSG-1186 genotypes was used for PCR amplification of TaSS-IIIa1D gene using gene specific primers, and using pJET1.2/blunt cloning vector, and then sequenced to detect the SNPs. Two homologs of the TaSS-III genes, TaSS-IIIa and TaSS-IIIb, were found on the plus strand of chromosome 1 (1A, 1B & 1D) and minus strand of chromosome 2 (2A, 2B &2D), respectively. All homeologous copies of the gene contained 16 exons. Out of which, 3rd was the largest (1698-2757 bp) and 2nd the smallest (64 bp). Besides, 15 introns were identified in the gene. Among which 1, 5, and 8 were longer (391-2906 bp, 447-910 bp, and 591-899 bp), and 6, 13, 14, and 15 were shorter (75-114 bp, 83-99 bp, 87-90 bp, and 81-118 bp) in size. Exon 1 and 3 of the homologous and homeologous copies of the genes exhibited maximum variation. Introns were found relatively more variable than the exons. The average length of the total intronic region of the genes was estimated slightly longer than that of its coding sequence. TaSS-IIIa1B, TaSS-IIIa1D, and TaSS-IIIb2A contained 3 splice variants, TaSS-IIIb2B & TaSS-IIIb2D contained 2 splice variants and TaSS-IIIa1A contained only a single transcript. Phylogenetic analysis showed that copy of the gene present on the 1st chromosome (1A, 1B & 1D) share maximum similarity with HvSS-IIIa followed by BdSS-IIIa, OsSS-IIIa, SbSS-IIIa, and ZmSS-IIIa whereas, TaSS-IIIb exhibited maximum similarity with OsSS-IIIb followed by ZmSS-IIIb and SbSS-IIIb respectively. Maximum dissimilarity for SS-III genes was found between monocots and dicots. For both TaSS-III genes, the sequences found on A and D genomes were more similar than that of the gene on the B genome. Domain analysis revealed that the glycosyltransferase (GT) domain was most conserved among all the domains. Three SBDs were found in each of the homeologous copies of TaSS-IIIa & TaSS-IIIb protein, wherein the positions of tryptophan amino acids were found conserved. Expression analysis of both copies showed that TaSS-IIIb expresses in the tissues viz. leaf, stem, root, spike, & grain and in much higher amounts than TaSS-IIIa. Whereas, TaSSIIIa expression was highly specific to endosperm in the grain. The expression of the TaSS-III genes reduces due to heat stress.
Sequencing of the products of direct PCR and indirect vector cloning showed that in-vitro amplification and in vivo amplification products have no variation. A total of 49 SNPs were identified in 10,529bp of the TSS-IIIa1D gene between the PBW-343 and KSG-1186 genotypes. Twenty-nine specific SNPs were identified in heat-sensitive genotype (PBW-343), and 20 specific SNPs were identified in the heat-tolerant genotype (KSG-1186). There were 14 intronic and 15 exonic SNPs contributing to 18 transitions and 9 transversions in the PWB-343 genotype, reflecting the transition bias. While in genotype KSG-1186, 9 transitions, 9 transversions, and two deletions are contributing to 6 intronic and 14 exonic SNPs showing no such bias. Maximum SNPs were detected in 3rd and 8th exons of PBW-343, whereas in genotype KSG-1186, only 3rd exon contained maximum SNPs. Exon 3 was found to be evolutionarily highly variable among all monocots and dicots taxa. Between PBW-343 and KSG-1186, 18 SNPs consisting of 11 transitions, and 7 transversions were found, reflecting the transition bias. Seven SNPs found associated with SBD-1, SBD-2 and SS-CD domains of the TaSS-IIIa1D protein. In SBD-1, one non-synonymous and one synonymous mutation were observed in both PBW-343 and KSG-1186; in SBD-2, one non-synonymous mutation was observed in KSG-1186 whereas, one non-synonymous and one synonymous mutation were observed in SSCD of PBW-343.