The degenerate primers were designed based on the conserved NBS-LRR motifs among the known disease-resistance genes. A fragment of about 500 bp was amplified from genomic DNA of sweet potato using the specifically des...The degenerate primers were designed based on the conserved NBS-LRR motifs among the known disease-resistance genes. A fragment of about 500 bp was amplified from genomic DNA of sweet potato using the specifically designed degenerate primers. After cloning and sequencing, 20 NBS-LRR type of disease-resistance gene analogue （RGAs） in sweet potato were observed. The deduced amino acid sequence of DNA fragment contains the conserved motifs of NBS-LRR type RGAs, such as P-loop, Kinase-2α, Kinase-3α and GLPL domain. The 20 RGAs could be sorted into two subclasses, namely TIR- NBS-LRR type and non-TIR-NBS-LRR type. Compared with the known resistance genes including N, L6 and M, the percentages of homologous amino acid sequence in 10 TIR-NBS-LRR range between 21% -44%. While other 10 non-TIR-NBS-LRR assume 15% -46% homology with the known resistance genes （Prf, RPM1, RPS2, etc. ）. Consequently the RGAs may further be used as molecular marker for screening the candidate disease-resistance genes in sweet potato.展开更多
Alternative splicing is a major contributor to genomic complexity and proteome diversity, yet the analysis of alternative splicing for the sequence containing nucleotide binding site and leucine-rich repeats （NBS-LRR...Alternative splicing is a major contributor to genomic complexity and proteome diversity, yet the analysis of alternative splicing for the sequence containing nucleotide binding site and leucine-rich repeats （NBS-LRR） domain has not been explored in rice （Oryza sativa L.）. Hidden Markov model （HMM） searches were performed for NBS-LRR domain. 875 NBS-LRR-encoding sequences were obtained from the Institute for Genomic Research （TIGR）. All of them were used to blast Knowledge-based Oryza Molecular Biological Encyclopaedia （KOME）, TIGR rice gene index （TGI）, and Universal Protein Resource （UniProt） to obtain homologous full-length cDNAs （FL-cDNAs）, tentative consensus sequences, and protein sequences. Alternative splicing events were detected from genomic alignment of FL-cDNAs, tentative consensus sequences, and protein sequences, which provide valuable information on splice variants of genes. These sequences were aligned to the corresponding BAC sequences using the Spidey and Sim4 programs and each of the proteins was aligned by tBLASTn. Of the 875 NBS-LRR sequences, 119 （13.6%） sequences had alternative splicing where multiple FL-cDNAs, TGI sequences and proteins corresponded to the same gene. 71 intron retention events, 20 exon skipping events, 16 alternative termination events, 25 alternative initiation events, 12 alternative 5＇ splicing events, and 16 alternative 3＇ splicing events were identified. Most of these alternative splices were supported by two or more transcripts. The data sets are available at http：//www.bioinfor.org. Furthermore, the bioinformatics analysis of splice boundaries showed that exon skipping and intron retention did not exhibit strong consensus. This implies a different regulation mechanism that guides the expression of splice isoforms. This article also presents the analysis of the effects of intron retention on proteins. The C-terminal regions of alternative proteins turned out to be more variable than the N-terminal regions. Finally, tissue distribution and protein localization of alternative splicing were explored. The largest categories of tissue distributions for alternative splicing were shoot and callus. More than one-thirds of protein localization for splice forms was plasma membrane and cytoplasm. All the NBS-LRR proteins for splice forms may have important function in disease resistance and activate downstream signaling pathways.展开更多
文摘The degenerate primers were designed based on the conserved NBS-LRR motifs among the known disease-resistance genes. A fragment of about 500 bp was amplified from genomic DNA of sweet potato using the specifically designed degenerate primers. After cloning and sequencing, 20 NBS-LRR type of disease-resistance gene analogue （RGAs） in sweet potato were observed. The deduced amino acid sequence of DNA fragment contains the conserved motifs of NBS-LRR type RGAs, such as P-loop, Kinase-2α, Kinase-3α and GLPL domain. The 20 RGAs could be sorted into two subclasses, namely TIR- NBS-LRR type and non-TIR-NBS-LRR type. Compared with the known resistance genes including N, L6 and M, the percentages of homologous amino acid sequence in 10 TIR-NBS-LRR range between 21% -44%. While other 10 non-TIR-NBS-LRR assume 15% -46% homology with the known resistance genes （Prf, RPM1, RPS2, etc. ）. Consequently the RGAs may further be used as molecular marker for screening the candidate disease-resistance genes in sweet potato.
基金This work was supported by Natural Sciences Foundation of Guangdong Province (No. 0409078)Natural Sciences Foundation from the Education Department of Guangdong Province (No. z02051).
文摘Alternative splicing is a major contributor to genomic complexity and proteome diversity, yet the analysis of alternative splicing for the sequence containing nucleotide binding site and leucine-rich repeats （NBS-LRR） domain has not been explored in rice （Oryza sativa L.）. Hidden Markov model （HMM） searches were performed for NBS-LRR domain. 875 NBS-LRR-encoding sequences were obtained from the Institute for Genomic Research （TIGR）. All of them were used to blast Knowledge-based Oryza Molecular Biological Encyclopaedia （KOME）, TIGR rice gene index （TGI）, and Universal Protein Resource （UniProt） to obtain homologous full-length cDNAs （FL-cDNAs）, tentative consensus sequences, and protein sequences. Alternative splicing events were detected from genomic alignment of FL-cDNAs, tentative consensus sequences, and protein sequences, which provide valuable information on splice variants of genes. These sequences were aligned to the corresponding BAC sequences using the Spidey and Sim4 programs and each of the proteins was aligned by tBLASTn. Of the 875 NBS-LRR sequences, 119 （13.6%） sequences had alternative splicing where multiple FL-cDNAs, TGI sequences and proteins corresponded to the same gene. 71 intron retention events, 20 exon skipping events, 16 alternative termination events, 25 alternative initiation events, 12 alternative 5＇ splicing events, and 16 alternative 3＇ splicing events were identified. Most of these alternative splices were supported by two or more transcripts. The data sets are available at http：//www.bioinfor.org. Furthermore, the bioinformatics analysis of splice boundaries showed that exon skipping and intron retention did not exhibit strong consensus. This implies a different regulation mechanism that guides the expression of splice isoforms. This article also presents the analysis of the effects of intron retention on proteins. The C-terminal regions of alternative proteins turned out to be more variable than the N-terminal regions. Finally, tissue distribution and protein localization of alternative splicing were explored. The largest categories of tissue distributions for alternative splicing were shoot and callus. More than one-thirds of protein localization for splice forms was plasma membrane and cytoplasm. All the NBS-LRR proteins for splice forms may have important function in disease resistance and activate downstream signaling pathways.
