Nucleotide sequence of cryIA gene cloned from Btk isolate of Bacillus thuringiensis and comparison with cryIA(c) gene from B. thuringiensis subsp. kenyae Sekuen nukleotida gen cryIA dari B.thuringiensis isolat Btk dibandingkan dengan gen crylA(c) dari B. thuringiensis subsp. Kenyae

Asmini BUDIANI, Djoko SANTOSO

Abstract


Ringkasan

 

Perakitan tanaman perkebunan yang toleran terhadap serangga hama dapat ditempuh melalui rekayasa genetika menggunakan gen cry. Gen cryIA merupakan gen cry yang paling banyak dipelajari di antara gen cry lainnya. Berdasarkan homology sekuen dan spesifisitas protein yang disandinya terhadap serangga sasaran, gen ini telah diklasifikasikan menjadi 10 subklas. Tulisan ini melaporkan hasil sekuensing (ragmen gen cryIA penyandi domain toksin yang diisolasi dengan teknik PCR dari Bacillus thuringiensis isolat Btk dan diklon menggunakan vektor pGEM­T. Untuk menentukan sekuen gen cryIA yang berukuran sekitar 2 kb tersebut, dilakukan kons­truksi satu seri mutan terdelesi searah dari ujung 5' menggunakan kit Erase-a-Base-System. Tiga DNA gen cryIA mutan dengan tingkat delesi yang sesuai dan satu nonmutan dipilih untuk sekuensing DNAnya. Sekuensing dilakukan dari satu arah menggunakan primer universal SP6 pada alat ABI 377A automatic DNA sequencer. Sekuen lengkap dari gen cryIA diperoleh dengan cara meng­gabungkan sekuen ketiga mutan dengan sekuen dari gen cryIA nonmutan secara manual. Untuk konfirmasi sekuen ujung 3', dilakukan sekuensing dari arah lainnya menggunakan primer universal T7. Sekuen lengkap dari fragmen tersebut mengandung 2021 nukleotida dan menyandi protein dengan 673 asam amino. Dibandingkan dengan sekuen gen crylA(c) dari B. thuringiensis subsp. kenyae, terlihat adanya sepuluh mutasi titik masing-masing pada nukleotida ke 444, 477, 1089, 1092, 1098, 1242, 1566, 1869, 1906 dan 1961. Tujuh mutasi titik pada nukleotida ke 444, 477, 1089, 1092, 1242, 1566, dan 1869 tidak merubah asam amino, sedangkan tiga mutasi lainnya mengakibatkan perubahan asam amino, yaitu pada nukleotida ke 1098 (kodon ke 366, yang menyebabkan perubahan dari Phe menjadi Leu), nukleotida ke 1906 (kodon ke 636, yang mengubah Val menjadi Leu) dan pada nukleotida ke 1961(kodon ke 654, yang mengubah Cys menjadi Tyr).


Summary

 

Estate crops tolerant to pests can be devel­opment through genetic engineering using cry gene. CryIA is the best studied among cry genes. Based on the sequence homology and specificity of their encoded proteins to the, targeted insect, these genes have been classified into 10 sub­classes. This paper reports sequencing of cryIA gene fragment en-coding toxin domain isolated from Btk isolates of Bacillus thuringiensis using PCR technique and cloned with pGEM-T vector. To determine the full sequence of the 2-kb gene fragment, a series of mutants uni-directionally deleted at the 5'-end were constructed. Mutation was done using Erase-a Base-System kit. Three DNA mutants with appropriate degree of deletion and the un-mutated DNA were chosen for sequencing. Sequencing was conducted from one direction with SP6 universal primer using the ABI 377A automatic DNA sequencer. The full sequence of cryIA fragment was assembled manually using the sequences of DNA mutants and the non-mutant cryIA fragment. To confirm the sequence of the 3'-end, sequencing from the other direction was performed using the T7 universal primer.The completed sequence of the fragment contains 2021 nucleotides encoding a protein of 673 amino acids. Compares to the sequence of cryIA(c) from B. thuringiensis subsp. kenyae, it was shown that there were ten point mutations (nucleotides of 444, 477, 1089, 1092, 1098, 1242, 1566, 1869, 1906 and 1961), sevent of them (nucleotides of 444, 477, 1089, 1092, 1242, 1566 and 1869) were identified as silent mutations, while the other three substituted the amino acids, which are at the nucleotide 1089 (codon 366, substitution of Leu for Phe), nucleotide 1906 (codon 636, substitution of Leu for Val), and nucleotide 1961 (codon 654, substitution of Tyr for Cys).



Keywords


cryIA(c) gene, Bacillus thuringiensis, DNA sequencing, uni-deleted mutant

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DOI: http://dx.doi.org/10.22302/iribb.jur.mp.v68i1.134

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