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Molecular characterization of genes encoding isoamylase-type debranching enzyme in tuberous root of sweet potato, Ipomoea batatas (L.) Lam.

ABSTRACT: Isoamylase (ISA) is a starch debranching enzyme that removes ?-1,6-glucosidic linkages in ?-polyglucans such as amylopectin. From previous studies, plant isoamylases have been shown to play a crucial role in amylopectin biosynthesis; however, little is known about their function in storage root tissues of plants such as cassava, yam and sweet potato. In this study, we isolated cDNA clones and characterized the cDNA nucleotide sequences of three genes (IbISA1, IbISA2, IbISA3) encoding isoamylase from sweet potato (Ipomoea batatas (L.) cv. White Star). Deduced amino acid sequences of the three isolated IbISAs have the specific regions that are highly conserved among the ?-amylase family members. The product of IbISA2 is predicted to be enzymatically inactive, like other plant ISA2s, due to replacement of amino acid residues that are important for hydrolytic reaction. qRT-PCR analysis demonstrated that expression of IbISA2 was higher than that of the other two IbISAs (IbISA1 and IbISA3) in tuberous root at 109 days after planting, at which stage of tuberous root was at which stage tuberous roots were almost fully developed almost developed. This expression pattern observed in our experiments was different from that in other sink organs, such as seeds (endosperms), indicating that orchestration of ISA gene expression may depend on the differences in sink organ type between tuberous roots and seeds. The molecular characterization of three IbISA genes and their expression analysis in this study will contribute to further studies on starch biosynthesis in sweet potato, especially in storage root.

SUBMITTER: Nabemoto M

PROVIDER: S-EPMC6587035 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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Molecular characterization of genes encoding isoamylase-type debranching enzyme in tuberous root of sweet potato, Ipomoea batatas (L.) Lam.

Nabemoto Moe M Watanabe Riho R Ohsu Mizuho M Sato Kaname K Otani Motoyasu M Nakayachi Osamu O Watanabe Masao M

Plant biotechnology (Tokyo, Japan) 20161126 5

Isoamylase (ISA) is a starch debranching enzyme that removes α-1,6-glucosidic linkages in α-polyglucans such as amylopectin. From previous studies, plant isoamylases have been shown to play a crucial role in amylopectin biosynthesis; however, little is known about their function in storage root tissues of plants such as cassava, yam and sweet potato. In this study, we isolated cDNA clones and characterized the cDNA nucleotide sequences of three genes (IbISA1, IbISA2, IbISA3) ...[more]

PMID: 31274996

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Project description:Background and aimsThe tuberization mechanism of sweet potato (Ipomoea batatas) has long been studied using various approaches. Morphological data have revealed that the tuberizing events result from the activation of the cambium, followed by cell proliferation. However, uncertainties still remain regarding the regulators participating in this signal-transduction pathway. An attempt was made to characterize the role of one MADS-box transcription factor, which was preferentially expressed in sweet potato roots at the early tuberization stage.MethodsA differential expression level of IbMADS1 (Ipomoea batatas MADS-box 1) was detected temporally and spatially in sweet potato tissues. IbMADS1 responses to tuberization-related hormones were assessed. In order to identify the evolutionary significance, the expression pattern of IbMADS1 was surveyed in two tuber-deficient Ipomoea relatives, I. leucantha and I. trifida, and compared with sweet potato. In functional analyses, potato (Solanum tuberosum) was employed as a heterologous model. The resulting tuber morphogenesis was examined anatomically in order to address the physiological function of IbMADS1, which should act similarly in sweet potato.Key resultsIbMADS1 was preferentially expressed as tuberous root development proceeded. Its expression was inducible by tuberization-related hormones, such as jasmonic acid and cytokinins. In situ hybridization data showed that IbMADS1 transcripts were specifically distributed around immature meristematic cells within the stele and lateral root primordia. Inter-species examination indicated that IbMADS1 expression was relatively active in sweet potato roots, but undetectable in tuber-deficient Ipomoea species. IbMADS1-transformed potatoes exhibited tuber morphogenesis in the fibrous roots. The partial swellings along fibrous roots were mainly due to anomalous proliferation and differentiation in the xylem.ConclusionsBased on this study, it is proposed that IbMADS1 is an important integrator at the initiation of tuberization. As a result, the initiation and development of tuberous roots seems to be well regulated by a network involving a MADS-box gene in which such hormones as jasmonic acid and cytokinins may act as trigger factors.

Project description:BACKGROUND: Sweet potato (Ipomoea batatas L. [Lam.]) ranks among the top six most important food crops in the world. It is widely grown throughout the world with high and stable yield, strong adaptability, rich nutrient content, and multiple uses. However, little is known about the molecular biology of this important non-model organism due to lack of genomic resources. Hence, studies based on high-throughput sequencing technologies are needed to get a comprehensive and integrated genomic resource and better understanding of gene expression patterns in different tissues and at various developmental stages. METHODOLOGY/PRINCIPAL FINDINGS: Illumina paired-end (PE) RNA-Sequencing was performed, and generated 48.7 million of 75 bp PE reads. These reads were de novo assembled into 128,052 transcripts (? 100 bp), which correspond to 41.1 million base pairs, by using a combined assembly strategy. Transcripts were annotated by Blast2GO and 51,763 transcripts got BLASTX hits, in which 39,677 transcripts have GO terms and 14,117 have ECs that are associated with 147 KEGG pathways. Furthermore, transcriptome differences of seven tissues were analyzed by using Illumina digital gene expression (DGE) tag profiling and numerous differentially and specifically expressed transcripts were identified. Moreover, the expression characteristics of genes involved in viral genomes, starch metabolism and potential stress tolerance and insect resistance were also identified. CONCLUSIONS/SIGNIFICANCE: The combined de novo transcriptome assembly strategy can be applied to other organisms whose reference genomes are not available. The data provided here represent the most comprehensive and integrated genomic resources for cloning and identifying genes of interest in sweet potato. Characterization of sweet potato transcriptome provides an effective tool for better understanding the molecular mechanisms of cellular processes including development of leaves and storage roots, tissue-specific gene expression, potential biotic and abiotic stress response in sweet potato.

Dataset Information

Molecular characterization of genes encoding isoamylase-type debranching enzyme in tuberous root of sweet potato, Ipomoea batatas (L.) Lam.

Publications

Molecular characterization of genes encoding isoamylase-type debranching enzyme in tuberous root of sweet potato, <i>Ipomoea batatas</i> (L.) Lam.

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