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Transcriptome wide identification and characterization of starch branching enzyme in finger millet.

ABSTRACT: Starch-branching enzymes (SBEs) are one of the four major enzyme classes involved in starch biosynthesis in plants and play an important role in determining the structure and physical properties of starch granules. Multiple SBEs are involved in starch biosynthesis in plants. Finger millet is calcium rich important serial crop belongs to grass family and the transcriptome data of developing spikes is available on NCBI. In this study it was try to find out the gene sequence of starch branching enzyme and annotate the sequence and submit the sequence for further use. Rice SBE sequence was taken as reference and for characterization of the sequence different in silico tools were used. Four domains were found in the finger millet Starch branching enzyme like alpha amylase catalytic domain from 925 to2172 with E value 0, N-terminal Early set domain from 634 to 915 with E value 1.62 e-42, Alpha amylase, C-terminal all-beta domain from 2224 to 2511 with E value 5.80e-24 and 1,4-alpha-glucan-branching enzyme from 421 to 2517 with E value 0. Major binding interactions with the GLC (alpha-d-glucose), CA (calcium ion), GOL (glycerol), TRS (2-amino-2-hydroxymethylpropane- 1, 3-diol), MG (magnesium ion) and FLC (citrate anion) are fond with different residues. It was found in the phylogenetic study of the finger millet SBE with the 6 species of grass family that two clusters were form A and B. In cluster A, finger millet showed closeness with Oryzasativa and Setariaitalica, Sorghum bicolour and Zea mays while cluster B was formed with Triticumaestivum and Brachypodium distachyon. The nucleotide sequence of Finger millet SBE was submitted to NCBI with the accession no KY648913 and protein structure of SBE of finger millet was also submitted in PMDB with the PMDB id - PM0080938. This research presents a comparative overview of Finger millet SBE and includes their properties, structural and functional characteristics, and recent developments on their post-translational regulation.

SUBMITTER: Tyagi R

PROVIDER: S-EPMC5512855 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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Transcriptome wide identification and characterization of starch branching enzyme in finger millet.

Tyagi Rajhans R Tiwari Apoorv A Garg Vijay Kumar VK Gupta Sanjay S

Bioinformation 20170630 6

Starch-branching enzymes (SBEs) are one of the four major enzyme classes involved in starch biosynthesis in plants and play an important role in determining the structure and physical properties of starch granules. Multiple SBEs are involved in starch biosynthesis in plants. Finger millet is calcium rich important serial crop belongs to grass family and the transcriptome data of developing spikes is available on NCBI. In this study it was try to find out the gene sequence of starch branching enz ...[more]

PMID: 28729759

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Project description:BackgroundStarch is stored in higher plants as granules composed of semi-crystalline amylopectin and amorphous amylose. Starch granules provide energy for the plant during dark periods and for germination of seeds and tubers. Dietary starch is also a highly glycemic carbohydrate being degraded to glucose and rapidly absorbed in the small intestine. But a portion of dietary starch, termed "resistant starch" (RS) escapes digestion and reaches the large intestine, where it is fermented by colonic bacteria producing short chain fatty acids (SCFA) which are linked to several health benefits. The RS is preferentially derived from amylose, which can be increased by suppressing amylopectin synthesis by silencing of starch branching enzymes (SBEs). However all the previous works attempting the production of high RS crops resulted in only partly increased amylose-content and/or significant yield loss.ResultsIn this study we invented a new method for silencing of multiple genes. Using a chimeric RNAi hairpin we simultaneously suppressed all genes coding for starch branching enzymes (SBE I, SBE IIa, SBE IIb) in barley (Hordeum vulgare L.), resulting in production of amylose-only starch granules in the endosperm. This trait was segregating 3:1. Amylose-only starch granules were irregularly shaped and showed peculiar thermal properties and crystallinity. Transgenic lines retained high-yield possibly due to a pleiotropic upregualtion of other starch biosynthetic genes compensating the SBEs loss. For gelatinized starch, a very high content of RS (65 %) was observed, which is 2.2-fold higher than control (29%). The amylose-only grains germinated with same frequency as control grains. However, initial growth was delayed in young plants.ConclusionsThis is the first time that pure amylose has been generated with high yield in a living organism. This was achieved by a new method of simultaneous suppression of the entire complement of genes encoding starch branching enzymes. We demonstrate that amylopectin is not essential for starch granule crystallinity and integrity. However the slower initial growth of shoots from amylose-only grains may be due to an important physiological role played by amylopectin ordered crystallinity for rapid starch remobilization explaining the broad conservation in the plant kingdom of the amylopectin structure.

Project description:BackgroundThe nucleotide binding site leucine rich repeat (NBLRR) genes significantly regulate defences against phytopathogens in plants. The genome-wide identification and analysis of NBLRR genes have been performed in several species. However, the detailed evolution, structure, expression of NBLRRs and functional response to Magnaporthe grisea are unknown in finger millet (Eleusine coracana (L.) Gaertn.).ResultsThe genome-wide scanning of the finger millet genome resulted in 116 NBLRR (EcNBLRRs1-116) encompassing 64 CC-NB-LRR, 47 NB-LRR and 5 CCR-NB-LRR types. The evolutionary studies among the NBLRRs of five Gramineae species, viz., purple false brome (Brachypodium distachyon (L.) P.Beauv.), finger millet (E. coracana), rice (Oryza sativa L.), sorghum (Sorghum bicolor L. (Moench)) and foxtail millet (Setaria italica (L.) P.Beauv.) showed the evolution of NBLRRs in the ancestral lineage of the target species and subsequent divergence through gene-loss events. The purifying selection (Ka/Ks < 1) shaped the expansions of NBLRRs paralogs in finger millet and orthologs among the target Gramineae species. The promoter sequence analysis showed various stress- and phytohormone-responsive cis-acting elements besides growth and development, indicating their potential role in disease defence and regulatory mechanisms. The expression analysis of 22 EcNBLRRs in the genotypes showing contrasting responses to Magnaporthe grisea infection revealed four and five EcNBLRRs in early and late infection stages, respectively. The six of these nine candidate EcNBLRRs proteins, viz., EcNBLRR21, EcNBLRR26, EcNBLRR30, EcNBLRR45, EcNBLRR55 and EcNBLRR76 showed CC, NB and LRR domains, whereas the EcNBLRR23, EcNBLRR32 and EcNBLRR83 showed NB and LRR somains.ConclusionThe identification and expression analysis of EcNBLRRs showed the role of EcNBLRR genes in assigning blast resistance in finger millet. These results pave the foundation for in-depth and targeted functional analysis of EcNBLRRs through genome editing and transgenic approaches.

Project description:Amylopectin, the major component of starch, is synthesized by synergistic activity of multiple isozymes of starch synthases (SSs) and branching enzymes (BEs). The frequency and length of amylopectin branches determine the functionality of starch. In the rice endosperm, BEIIb generates short side chains of amylopectin and SSI elongates those branches, which can be further elongated by SSIIa. Absence of these enzymes greatly affects amylopectin structure. SSI, SSIIa, and BEIIb associate with each other and with other starch biosynthetic enzymes although SSIIa is low activity in japonica rice. The aim of the current study was to understand how the activity of starch biosynthetic enzyme complexes is compensated in the absence of SSI or BEIIb, and whether the compensatory effects are different in the absence of BEIIb or in the presence of inactive BEIIb. Interactions between starch biosynthetic enzymes were analyzed using one ss1 null mutant and two be2b japonica rice mutants (a mutant producing inactive BEIIb and a mutant that did not produce BEIIb). Soluble proteins extracted from the developing rice seeds were separated by gel filtration chromatography. In the absence of BEIIb activity, BEIIa was eluted in a broad molecular weight range (60-700 kDa). BEIIa in the wild-type was eluted with a mass below 300 kDa. Further, majority of inactive BEIIb co-eluted with SSI, SSIIa, and BEI, in a mass fraction over 700 kDa, whereas only small amounts of these isozymes were found in the wild-type. Compared with the be2b lines, the ss1 mutant showed subtle differences in protein profiles, but the amounts of SSIIa, SSIVb, and BEI in the over-700-kDa fraction were elevated. Immunoprecipitation revealed reduced association of SSIIa and BEIIb in the ss1 mutant, while the association of BEIIb with SSI, SSIIa, SSIVb, BEI, and BEIIa were more pronounced in the be2b mutant that produced inactive BEIIb enzyme. Mass spectrometry and western blotting revealed that SSI, SSIIa, SSIIIa, BEI, BEIIa, starch phosphorylase 1, and pullulanase were bound to the starch granules in the be2b mutants, but not in the wild-type and ss1 mutant. These results will aid the understanding of the mechanism of amylopectin biosynthesis.

Project description:BackgroundIn finger millet, calcium is one of the important and abundant mineral elements. The molecular mechanisms involved in calcium accumulation in plants remains poorly understood. Transcriptome sequencing of genetically diverse genotypes of finger millet differing in grain calcium content will help in understanding the trait.Principal findingIn this study, the transcriptome sequencing of spike tissues of two genotypes of finger millet differing in their grain calcium content, were performed for the first time. Out of 109,218 contigs, 78 contigs in case of GP-1 (Low Ca genotype) and out of 120,130 contigs 76 contigs in case of GP-45 (High Ca genotype), were identified as calcium sensor genes. Through in silico analysis all 82 unique calcium sensor genes were classified into eight calcium sensor gene family viz., CaM & CaMLs, CBLs, CIPKs, CRKs, PEPRKs, CDPKs, CaMKs and CCaMK. Out of 82 genes, 12 were found diverse from the rice orthologs. The differential expression analysis on the basis of FPKM value resulted in 24 genes highly expressed in GP-45 and 11 genes highly expressed in GP-1. Ten of the 35 differentially expressed genes could be assigned to three documented pathways involved mainly in stress responses. Furthermore, validation of selected calcium sensor responder genes was also performed by qPCR, in developing spikes of both genotypes grown on different concentration of exogenous calcium.ConclusionThrough de novo transcriptome data assembly and analysis, we reported the comprehensive identification and functional characterization of calcium sensor gene family. The calcium sensor gene family identified and characterized in this study will facilitate in understanding the molecular basis of calcium accumulation and development of calcium biofortified crops. Moreover, this study also supported that identification and characterization of gene family through Illumina paired-end sequencing is a potential tool for generating the genomic information of gene family in non-model species.

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Transcriptome wide identification and characterization of starch branching enzyme in finger millet.

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Transcriptome wide identification and characterization of starch branching enzyme in finger millet.

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