PCR cloning of the full-length cDNA for the seed protein canavalin from the jack bean plant, Canavalis ensiformis.
PCR cloning of the full-length cDNA for the seed protein canavalin from the jack bean plant, Canavalis ensiformis.
复制标题
PCR 克隆来自刀豆植物 Canavalis ensiformis 的种子蛋白刀豆蛋白的全长 cDNA。
DOI：
10.1007/bf00018469
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发表时间：
1992
影响因子：
5.1
通讯作者：
McPherson,A
中科院分区：
文献类型：
--
作者：
Ng,JD;Stinchcombe,T;Ko,TP;Alexander,E;McPherson,A
Canavalin is the major storage protein in jack bean (Canavalia ensiformis) belonging to the classical vicilin fraction. To obtain detailed genetic and structural information on the reserve proteins of legumes a full-length eDNA for canavalin was generated by use of the polymerase chain reaction (PCR)[1]. A single-stranded eDNA template synthesized from total jack bean RNA by reverse transcriptase [2] was used with primer pairs selected from the 5'and 3'sequence of the eDNA coding for the vicilin protein from Canavalia gladiata [3]. The primers were 5'primer 5'-ATCATCCCCTCACACTGCAATACCA-3'and the 3'primer 5'-AGAGAGAAAAGAAAACCATGATGGT-3'. The reaction conditions included the standard PCR buffer [1] using 25 pmol of each primer in a 100# 1 volume. PCR was carried out using a DNA Thermal Cycler (Ericomp) for 90 s at 96 C initial denaturation, followed by 30 cycles for 30 s at 55 C, 2 min at 72 C, 15 s at 96 C. A final extension step was performed for 2 min at 55 o C and 7 min at 72 o C. The fragment obtained was directly sequenced by S anger's dideoxy method [4] with the Sequenase kit version 2.0 (United States Biochemical, Cleveland, OH) following the directions supplied by the manufacturer with slight modifications. The direct sequencing of the PCR-cloned eDNA shows a very similar nucleotide and deduced amino acid sequence to the vicilin protein isolated from C. gladiata seeds [3]. Only 5 nucleotides differ: C to G at base 505, C to T at base 889, C to A at base 1058, A to C at base 1166 and A to T at base 1213. There are two base changes that are silent while the other three generate the amino acid substitutions; asparagine to lysine at residue 161 (AAC to AAG), leucine to isoleucine at residue 346 (CTC to ATC) and asparagine to histidine at residue 382 (AAC to CAC). We previously demonstrated by amino acid sequence homology searches using C. gladiata that canavalin contains an internal sequence homology which indicates that its gene was evolved by duplication and mutation of an ancestral genetic domain [5]. We further showed by comparison with other seed reserve proteins whose sequences were known, that this gene structure is a common feature of all of the vicilin class proteins. In addition, this same ancestral genetic domain was found to comprise a portion of the legumin class proteins as well [5].The internal amino acid sequence homology is indicated in Fig. 1 by double underscoring. The two homologous segments, found in the amino terminal half and the carboxyl terminal half of the protein, have been shown by X-ray diffraction analysis of the three-dimensional structure of the protein to fold into compact eight-stranded anti-
