Isolation and sequence analysis of the genomic DNA fragment encoding an aspartic proteinase inhibitor homologue from potato (Solanum tuberosum L.)

Summary Cacao is socially and economically an important commodity for Indonesia, in which the cacao plantations have been challenged with a threatening pest, cacao pod borer (CPB). This research aimed to identify and clone PIN (proteinase inhibitor), a gene carrying resistance of plant to some chewing pests like CPB. The methodology included several experiments. Detection of PIN in cacao was done by PCR using PIN-specific heterologous primers and cacao genomic DNA as templates. Cloning vector pGEM-T was utilized to clone the PCR products. Sequence analysis was conducted with BlastX and Blast Special programs from NCBI. Alignment analysis to determine genetic similarity was performed with ClustalW from EBI. Thirteen of the 18 clones tested were detected to have PIN homologs. Two DNA fragments from cacao clones putatively tolerant to CPB, MJ-1 and LW-1, were sequenced. One of them, MJ-1 was cloned. Sequence analyses of the fragments of both cacao clones, indicated that they have PIN homologs and a very closed genetic relation with 96% level of similarity. Ringkasan Kakao adalah komoditas yang secara sosial maupun ekonomi penting bagi Indonesia, dimana perkebunan kakao menghadapi masalah serius hamapenggerek buah kakao (PBK). Penelitian ini bertujuan mengidentifikasi dan mengklon PIN (inhibitor proteinase), gen yang membawa sifat ketahanan tanaman terhadap hama ulat seperti PBK. Metodologinya terdiri dari beberapa percobaan. Deteksi PIN di dalam kakao dikerjakan dengan PCR menggunakan primer heterologous yang spesifik terhadap PIN dan DNA genomik kakao sebagai templetnya. Vektor kloning pGEM-T digunakan untuk mengklon produk PCR. Analisis sekuen dilakukan dengan program BlastX dan Blast spesial dari NCBI. Analisis penjajaran (alignment) untuk menentukan kemiripan genetik menggunakan program ClustalW dari EBI. Tiga belas dari 18 klon kakao yang diuji menunjukkan adanya homolog PIN. Dua DNA fragmen dari klon harapan tahan, MJ-1 dan LW-1, telah ditentukan sekuen nukleotidanya. Satu diantara-nya, MJ-1 berhasil diklon. Analisis sekuen kedua klon tersebut menunjukkan identitas sebagai homolog PIN dan keduanya memiliki kemiripan genetik yang tinggi.

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Cloning and sequence analysis of the genomic DNA fragment encoding oryzacystatin

Gene ◽

10.1016/0378-1119(89)90186-8 ◽

1989 ◽

Vol 81 (2) ◽

pp. 259-265 ◽

Cited By ~ 21

Author(s):

Kendo Hiroto ◽

Emori Yasufumi ◽

Abe Keiko ◽

Suzuki Koichi ◽

Arai Soichi

Keyword(s):

Sequence Analysis ◽

Genomic Dna ◽

Dna Fragment

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A Kunitz-type proteinase inhibitor with differential solubility during the development of potato (Solanum tuberosum L.) tubers

Potato Research ◽

10.1007/bf02358468 ◽

1996 ◽

Vol 39 (4) ◽

pp. 493-505

Author(s):

Cristina Mitsumori ◽

K. Yamagishi ◽

T. Itoh ◽

Y. Kikuta

Keyword(s):

Solanum Tuberosum ◽

Proteinase Inhibitor ◽

Solanum Tuberosum L ◽

Kunitz Type ◽

Differential Solubility

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Deteksi dan analisis sekuen gen inhibitor proteinase pada beberapa klon kakao harapan tahan penggerek buah kakao dari Sulawesi Selatan Detection and sequence analysis of proteinase inhibitor gene in cacao clones putatively cacao pod borer-tolerant from South Sulawesi

E-Journal Menara Perkebunan ◽

10.22302/iribb.jur.mp.v72i1.124 ◽

2016 ◽

Vol 72 (1) ◽

Author(s):

Abdul Mollah S. JAYA ◽

Hajrial ASWIDINNOOR ◽

Djoko SANTOSO

Keyword(s):

Sequence Analysis ◽

Proteinase Inhibitor ◽

Genomic Dna ◽

Genetic Similarity ◽

Sequence Analyses ◽

South Sulawesi ◽

Pod Borer ◽

Special Programs ◽

Pcr Products ◽

Alignment Analysis

Summary Cacao is socially and economically an important commodity for Indonesia, in which the cacao plantations have been challenged with a threatening pest, cacao pod borer (CPB). This research aimed to identify and clone PIN (proteinase inhibitor), a gene carrying resistance of plant to some chewing pests like CPB. The methodology included several experiments. Detection of PIN in cacao was done by PCR using PIN-specific heterologous primers and cacao genomic DNA as templates. Cloning vector pGEM-T was utilized to clone the PCR products. Sequence analysis was conducted with BlastX and Blast Special programs from NCBI. Alignment analysis to determine genetic similarity was performed with ClustalW from EBI. Thirteen of the 18 clones tested were detected to have PIN homologs. Two DNA fragments from cacao clones putatively tolerant to CPB, MJ-1 and LW-1, were sequenced. One of them, MJ-1 was cloned. Sequence analyses of the fragments of both cacao clones, indicated that they have PIN homologs and a very closed genetic relation with 96% level of similarity. Ringkasan Kakao adalah komoditas yang secara sosial maupun ekonomi penting bagi Indonesia, dimana perkebunan kakao menghadapi masalah serius hamapenggerek buah kakao (PBK). Penelitian ini bertujuan mengidentifikasi dan mengklon PIN (inhibitor proteinase), gen yang membawa sifat ketahanan tanaman terhadap hama ulat seperti PBK. Metodologinya terdiri dari beberapa percobaan. Deteksi PIN di dalam kakao dikerjakan dengan PCR menggunakan primer heterologous yang spesifik terhadap PIN dan DNA genomik kakao sebagai templetnya. Vektor kloning pGEM-T digunakan untuk mengklon produk PCR. Analisis sekuen dilakukan dengan program BlastX dan Blast spesial dari NCBI. Analisis penjajaran (alignment) untuk menentukan kemiripan genetik menggunakan program ClustalW dari EBI. Tiga belas dari 18 klon kakao yang diuji menunjukkan adanya homolog PIN. Dua DNA fragmen dari klon harapan tahan, MJ-1 dan LW-1, telah ditentukan sekuen nukleotidanya. Satu diantara-nya, MJ-1 berhasil diklon. Analisis sekuen kedua klon tersebut menunjukkan identitas sebagai homolog PIN dan keduanya memiliki kemiripan genetik yang tinggi.

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