Biochemical and molecular analyses of tuber-associated proteins in Cassava (Manihot esculentum Crantz)
2005
Azucena, V.A.
A molecular approach to study the tuber-associated proteins in cassava (Manihot esculenta Crantz) was conducted. Total soluble proteins were extracted using 200 mM Tris-HCl pH 8.2 and highest yield was obtained with the middle cylindrical/transverse sections of the tubers. Osborne fractionation scheme revealed globulins and prolamines as the major tuber proteins. A non-glycosylated 40 kDa globulin protein with pI of 6.5 was isolated and purified. The protein is rich in glycine, alanine and serine. It has an N-terminal sequence of DINGQXN and is moderately digestible on IVPD [in vitro protein digestibility]. The ratio of essential to total amino acids is 35.57% while the predicted PER is 2.14. Six of the essential amino acids were comparable with the FAO/WHO reference protein. PCR [polymerase chain reaction] and RT-PCR of gDNA and total RNA from cassava leaves and tuber generated a 600 bp DNA fragment. Based on sequence analysis, two different p roteins (CSV1 and CSV2) were derived from two genomic clues. CSV1 is approximately 187 amino acids of 20.6-21.5 kDa molecular mass of 19.5 kDa. Phylogenetic analyses showed that CSV1 and CSV2 are more related to patatin than dioscorin, sporamin and tarin. The deduced amino acid composition showed that both are highly charged basic proteins (pIs of 9.8 to 11.6) with biphasis properties. Two highly conserved domains (LSGRQ and WISAEFAL) were identified in both CSV1 and CSV2. based on BLAST search of the DNA and amino acid sequences, both CSV1 and CSV2 are novel proteins with no significant counterparts in the Genebank database.
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