Kloning dan karakterisasi gen penyandi inhibitor proteinase dari kulit buah kakao Cloning and characterization of gene encoding proteinase inhibitor of cacao pod wall

Mayta Novaliza ISDA, Musliar KASIM, . MANSYURDIN, Tetty CHAIDAMSARI, Djoko SANTOSO

Abstract


Summary

 

Attempts to increase cocoa production in Indonesia have been hinderred by attack of CPB (Conopomorpha cramerella). There has been no effective measures to control this pest leading to development of cacao planting materials which resistant to the pod borer. One of genes functioning in plant defense system against insect pests such as catepilar is Proteinase Inhibitor (PIN). This research aimed to isolate and characterize TcPIN gene of cacao pod wall. A clone of TcPIN was isolated with RT-PCR technique using total RNA of cacao pod wall and DNA primer designed based on the sequence Trypsin Inhibitor of cocoa bean accessible online. BlastX analysis of the sequence of the cDNA clone demonstrated that the ± 600 bp gene cloned with pGEM-T was PIN gene as indicated by highly homologous to Trypsin Inhibitor of Theobroma microcarpum resulted in 248 Score bits and E value 1 e-64. Two sequence alligment with the putative 21 kDa PIN  of cacao seed indicated a moderately high homology. Contrasting these two sequences however found some non identical amino acids implying some variations. 


Ringkasan

 

Usaha peningkatan produksi kakao di Indonesia terkendala antara lain oleh adanya serangan hama PBK (Conopomorpha cramerella). Untuk menanggulangi serangan PBK tersebut perlu adanya satu cara pengendalian yang efektif dan efisien, sehingga dapat mendorong usaha pengembangan bahan tanam yang tahan PBK. Salah satu gen  membawa sifat ketahanan tanaman terhadap hama ulat adalah Proteinase Inhibitor (PIN). Penelitian ini bertujuan untuk mengisolasi dan mengkarakterisasi gen TcPIN dari kulit buah kakao. Klon cDNA TcPIN diisolasi dari kulit buah kakao dengan teknik RT-PCR meng-gunakan RNA total kulit buah kakao dan primer DNA yang dirancang atas dasar sekuen Inhibitor Tripsin biji kakao yang diakses lewat internet.  Hasil analisis BlastX dari sekuen klon cDNA menunjukkan  bahwa gen berukuran  ± 600 pb yang telah diklon dengan pGEM-T tersebut adalah PIN karena memiliki homologi yang tinggi terhadap 21 kDa trypsin inhibitor dari Theobroma microcarpum yang meng-hasilkan Skor 248 bits dengan Nilai E 1e-64. Penjajaran dua sekuen dengan PIN putatif 21 kDa yang berasal dari biji kakao menunjuk-kan tingkat homologi yang tinggi, dengan perbedaan nyata sehingga dapat terlihat bahwa keduanya tidak identik.


Keywords


Theobroma cacao L, TcPIN gene, cDNA clone, cocoa-pod borer

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DOI: http://dx.doi.org/10.22302/iribb.jur.mp.v76i2.84

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