Construction of full-length pKT9FL clone encodes for glutelin from oil palm.

Abstract

Seed storage protein is classified into three classes, which are globulin, prolamin and glutelin. The protein was utilized during post-germinative period of plant growth. Three different isoforms of glutelin genes were isolated from oil palm kernel (Elais guineesis, Tenera) in previous research. One of the isoforms consists of two separate fragments, the 3'-end-fragment (900 bp) isolated from cDNA library and the 5'-end fragment (820 bp) isolated by using 5 '-RACE method. The full length of this clone is 1621 bp. Thus, this study was aimed to construct a complete full-length sequence of this oil palm glutelin cDNA by joining the two fragments at the overlapping sequence. The 5'-end fragment was first cloned into EcoRI and Smal site of pUC19 vector. The 3 '-end fragment was generated by PCR and joined with the 5'-end fragment at the Hpal site located at nt. 894 of the glutelin gene. The positive recombinant clone was selected by colony PCR technique and named as pKT9FL. The orientation and the joining site of Hpal in clone pKT9FL were verified by DNA sequencing. This complete sequence of clone pKT9FL consists of 1621 bp that encodes a polypeptide of 469 amino acid. Its 5'and 3'­ untranslated region is 52 bp and 152 bp respectively.