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Isolation and characterization of adp-glucose pyrophosphorylase enzyme in high and low starch variety of cassava
Cassava is an important root crop apart from the dietary aspect cassava demanded in diverse industrial application. It is an abundant source of starch. A key regulatory enzyme in the starch biosynthesis enzyme is AGPase. Starch accumulation is directly proportional to AGPase enzyme activity. Charact...
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| Muut tekijät: | |
| Aineistotyyppi: | Ph.D Thesis |
| Julkaistu: |
Vellayani
Department of Plant Biotechnology, College of Agriculture
2017
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| Aiheet: |
| Yhteenveto: | Cassava is an important root crop apart from the dietary aspect cassava demanded in diverse industrial application. It is an abundant source of starch. A key regulatory enzyme in the starch biosynthesis enzyme is AGPase. Starch accumulation is directly proportional to AGPase enzyme activity. Characterisation of AGPase gene opens to the world of crop improvement and production of high starch variety.
The study entitled “Isolation and characterization of ADP-glucose pyrophosphorylase (AGPase) enzyme in high and low starch variety of cassava” was carried out at the Division of Crop Improvement, ICAR-Central Tuber Crops Research Institute, Sreekariyam, Thiruvananthapuram during 2016-2017. The objective of the study was to analyse AGPase gene sequence variations in high starch cassava line 9S-127 and low starch cassava variety MNga-1. The gene amplification of AGPase is carried out with candidate primer.
From the Cassava improvement programme of ICAR-CTCRI, two varieties were selected for the study, high starch variety 9S-127 and low starch variety MNga-1. For the Amplification of AGPase, gene specific primers were designed using Primer 3 plus Bioinformatics software from the conserved regions of AGPase collected from NCBI. For the amplification of gene RNA isolated from high starch 9S-127 and low starch MNga-1 with manual and kit method were used. Manual method gives very poor quality and quantity of RNA. Kit method gave good yield of RNA. RNA was then converted into cDNA using reverse transcriptase enzyme. The gene specific primers were used for gene amplification. Sixteen primers were screened and three of them gave clear and reproducible bands in 1.2% agarose gel. Three primers AGPS1b, AGPS2d, AGPL showed bands in the size of 423bp, 581bp, 512bp. These specific bands were excised from the gel and purified with QIAquick Column. Purified gene fragments were used for sequencing in genetic analyser.
The sequences were compared with nucleotide data base in NCBI. This revealed that our amplified sequence showed similarity with AGPase sequence of Manihot esculenta. The sequences of high starch and low starch variety were compared using Clustal omega. After the analysis it was found that between MNga-1 and 9S-127 there is a difference in six base pairs. These sequences were in-vitro translated using ExPASy translate tool in bioinformatics. Among the translated amino acid functional amino acid were identified through BLAST-P bioinformatics tool. Each functional amino acid sequence developed from amplified sequences of MNga-1 and 9S-127 using three corresponding primer were compared using Clustal omega bioinformatics software. From the analysed data four amino acid alteration in large subunit and small subunit of MNga-1 and 9S-127 were identified.
. Sequence variation in small subunit of MNga-1 were methionine at 296th position is replaced by leucine, at 298th position tyrosine is replaced by phenylalanine, at 263rd position Arginine is replaced by serine and at 157th glutamic acid is replaced by glycine were identified. Methionine and leucine are highly similar and shows same property. Similarly, tyrosine and phenylalanine are highly similar and have same property.
In case of large subunit MNga-1at 137th position iso-leucine is replaced by phenylalanine, at 159th Iso-leucine is replaced by leucine, 169th arginine is replaced by alanine and at 181th threonine is replaced by asparagine. Iso-leucine and phenylalanine are highly similar. Iso-leucine and leucine have similar properties. Change in amino acid sequence leads to the change in conformation of the enzyme that will affect the enzyme activity. |
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| Ulkoasu: | 66p. |