Biotechnologie und Biomaterialien

Abstrakt

Isolation of Nucleotide Binding Site (NBS)-Leucine Rich Repeat (LRR) Resistant Gene Analogs (Rgas) In Arabica Coffee (Coffea Arabica L. Cv S.288)

Deepak Kumar and H.L. Sreenath

Cloning of resistance gene analogues against diverse pathogens from variety of plants in last decade has revealed that many of them share high level of conserved sequence motifs. The conserved backbone of amino acid motifs present in Nucleotide Binding Site (NBS) domain makes it possible to isolate resistance gene analogues by Polymerase chain reaction (PCR) with degenerate primers. Oligo-nucleotide primers combinations that target conserve motif of NBS domain as mentioned in earlier studies were used to amplify resistance gene analogues from Coffea arabica (S.288). PCR product amplified from genomic DNA as well as cDNA were cloned and sequenced. In the present study amplified resistance gene analogues from C.arabica genomic DNA and cDNA using Ploop-cof and GLPL-cof primers were cloned, sequenced using T7 / SP6 primers, analyzed at NCBI/SGN Nucleotide Data Bank. Analysis of these RGA leads to the understanding that difference in expression profile might be due to the difference present at sequence level of Resistant Gene Analogs (RGA) isolated from DNA and cDNA. Analysis also revealed presence of high level similarity at their sequences. Seven RGA isolated from genomic DNA of S.288 using non-degenerate primers, eleven more RGA were isolated from S.288 genomic DNA with degenerate oligo-nucleotide primers and thirty two RGA isolated from cDNA of S.288. Fifteen RGA clones isolated from cDNA prepared from rust race I infected leaf sample for 24 hours. BLASTN result showed these C.arabica RGA has a high level of similarity with C.canephora RGA. This confirm the integrity maintained among RGA even it was isolated from different coffee variety which has difference at there genome (C.arabica 2n= 44 and C.canephora 2n= 22). RGA isolated which has below 475 bp or above 530 bp in size along with both primer sequences in their end has either no match with any RGA or they match with microsatellite. There is one independent sequence from genomic DNA and four independent sequences from cDNA which has not given any BLASTN result, these sequences has primer sequence with them that indicates these may be belong to new type of RGA. The RGA reported in current study are mainly from the class A type of RGA.