Molecular cloning of cystatin genes from tomato leaves (Lycopersicon esculentum)

• Main Authors: Ya-Fen Hong, 洪雅芬
• Other Authors: 江善宗 吳如雯
• Format: Others
• Language: zh-TW
• Published: 2006
• Online Access: http://ndltd.ncl.edu.tw/handle/77724289825770239698

碩士 === 國立臺灣海洋大學 === 食品科學系 === 94 === Cystatins are inhibitors of cysteine proteases for defensive strategies against insect, fungi attack or mechanical wounding. Some plant cystatins possess all the characteristics of animal cystatin superfamily, including lack of disulfide bonds or cysteine residues like the stefin, high degree of homology with the member of cystatin and containing repeat cystatin-like domains similar to the kininogen. The genomic DNA sequences for potato cystatin (PMC) have been characterized. However a gene coding for tomato cystatin (TMC) which posseses similar biochemical properties to PMC has not been investigated. This study focuses on molecular cloning and characterization of cystatin genes from tomato leaves (Lycopersicon esculentum). The genomic DNA of tomato cystatin was isolated from tomato leaves UC204A. The genomic library of tomato cystatin was constructed by genomic PCR with the primers of LeCPIgs1 and LeCPIas2 designed from TMC gene sequences. In addition, genomic walking from UC204A genomic libraries is investigated using gene-specific primers LeCPIas4, LeCPIas5 designed from TMC gene sequences. The results showed that one intron occurs in each of tomato cystain-coding sequences, e.g. 78 and 102 bp in TMC domain 7 and 8, separately. A intron in each of tomato cystatin-like domains is located at the same region between ��-helix (LARFAVQDYNKXX) and catalytic QxVxG motif. A complete TMC-1 (tomato multicystatin domain 1) and partially TMC-2 are acquired by using the primers designed from PMC 5’ untranslation region and TMC 5’ end of DNA sequence. When compared with TMC and PMC introns, demonstrates the intron-1 in TMC and PMC are both not a multiple of three. TMC-1, TMC-2 and PMC-1, PMC-2 have high homology of 82% and 77% amino acid identitiy, separately.TMC-1 and TMC-6, 7 and 8 has low homology of 59-66% amino acid identity. TMC displayed a conserved region LARFAV, a catalytic QxVxG motif, Gly3, Gly4 in the N-terminal region and Trp77 in the C-terminal region, which are similar to other plant cystatins. The front TMC-1 sequence of the QxVxG motif (QKNGSSLEFEKVLKVKK) is noticeable differently with published TMC 6, 7, 8 domains (AHLEYVENLNVKE). A phylogenetic analysis demonstrated that TMC-1 and TMC-2 are closer to PMC-1 and PMC-2 compared to TMC 6, 7, and 8 domains. The structure of TMC-1 is analogous to several plant cystatins analyzed by Swiss-model system. In conclusion, it is shown high similarity of TMC and PMC in the DNA sequence with exons and introns.