| Cloning of plant resistance gene is greatly helpful to crop resistance breeding and the insight of resistance mechanism. Sweet potato (Ipomoa batatas Lam.) genome is extremely large, and contains many repetitive DNA sequences. That makes it seemingly laborious to use classic approach to clone the genes from sweet potato. So far, more than 60 genes resistant to different pathogens, including bacteria, fungi, nematodes and viruses, have been cloned from more than 10 plants, but no functional resistance gene has been separated from sweet potato. All of these plant disease resistance (R) genes have the conserved NBS and LRR domain. The existence of conserved domains provides opportunities for disease-resistance gene from sweet potato.Besides R gene, NPR1 is a key regulator of SAR (systemic acquired resistance) and ISR (induced systemic resistance), and plays an important role in basic resistance and the response mediated by R (resistance) gene. Overexpression of NPR1 can enhance resistance to multi-diseases. It's reported that NPR1 and its homolog genes exist in some important crops and confer broad-spectrum resistance. Defense response mediated by NPR1 is conserved in many plants. So, isolation of NPR1 and its homolog genes from plant will be the important significance in genetic engineering for disease-resistance.The major research contents of this dissertation included: (1) Degenerate primers based on conserved motif (P-loop and GLPL) of the nucleotide binding site (NBS) region from the cloned plant resistant-disease genes were used to isolate resistant gene analogues (RGA) from the genomes of Qingnong no.2 (high resistance to knot nematode) and Ipomoea trifdia (relative to sweetpotato) by polymerase chain reaction (PCR)-based approach. The sequence characterization and diversity analysis of theese RGAs are reported as well as their relationships with the NBS sequences of known R-genes from other plant plant species. These data permit insights into the origin, diversification and evolution of NBS-LRR resistance genes in Ipomoea genus.(2) Based on RGAs from sweet potato, the full length cDNA of NBS-LRR gene will be isolated with RT-PCR and RACE (Rapid amplification cDNA end). Southen blotting and transcription and expression analysis will be done.(3) Based on NPR1 gene from others plant, NPR1 in sweet potato will been isolated with RT-PCR and RACE. Southen blotting and transcription and expression analysis will also be done. The main experimental results were as follows:1．It is an effective way to clone gene using PCR with the primers designed on the basis of conserved motifs of cloned resistance genes. In this work, specific primers sequences were designed on the basis of conserved motifs (P-loop and GLPL) of cloned resistance genes, and subsequently were used to amplify the resistance gene analogues (RGA) from sweet potato cultivr Qingnong no.2 with thecharacter of knot nematodet resistance and Ipomoea trifida . 28 RGAs with uninterrupted open reading frames (ORFs) ( 22 RGAs origin from sweet potato, named IbRGA1-IbRGA22; 6 RGAs origin from Ipomoea trifida, named ItRGA1-It RGA6 ) were obtained. They were submitted to GenBank, and the accession number were DQ251184, DQ272296, DQ272297, DQ303440-DQ303442, DQ341401-DQ341403, DQ341405-DQ341408, DQ377949-DQ377957, DQ849027-DQ849032, respectively. The sequence analysis showed putative amino sequences from 28 RGAs contain the same conserved motifs such as P-loop, Kinase-2, Kinase-3a and HD presented in known NBS-LRR resistance genes from other plants and belong to NBS-LRR resistance-like proteins. Some RGAs from sweet potato share high similarity with Grol-4 ( from potato), Mi-I,Mi-2, Hero (from tomato), PPM1 (from Arabidopsis) . Sequence identity among the 28 RGA nucleotide sequences ranged from 35．5% to 99．4%, while identity of the deduced amino acid sequences from 28 RGAs ranged from 13．8% to 100%). The phylogenetic analyses for RGA nucleotide sequences and the deduced amino acids showed that RGAs from sweet potato were divided into two classed, TIR ( Drosophila Toll or human interleukin receptor-like ) type and nonTIR type. The analysis of codon composion, usage and Ka/Ks showed NBS-LRR type RGAs isolated from sweet potato might have the same mechanism of origin and evolution as same as that of other plants.2．Based on the sequence of one of resistance gene homologues IbRGA3, gene specific primers were designed, and the complete sequence of sweet potato resistance-related gene through the 5' RACE and 3' RACE methods were obtained. The resistance-related gene was named SPR1．The length of SPR1 was 3200 bp which included a 2667 bp-by complete open reading frame encoding SPRl protein of 888 amino acids. The deduced 888 amino acids SPRl protein consisted of nucleotide binding site (NBS) domain, a leucine-rich repeats(LRR) domain, and a hydrophobic domain, which were the conserved domains of plant resistance genes. The SPR1-related sequences were presented as 2-4 members in sweet potato genome according to the result of southern blotting. The SPR1 gene was constitutive gene with in sweet potato genome. The transcripts of SPR1 gene were detected in roots, stem and leaves of sweet potato respectively. Due to these features the SPRl gene was a member of nonTIR-NBS-LRR type resistance gene. The product of SPR1 was resistance-related protein which was similar to Arabdropsis RPM1 in the structure. The gene has been submitted to the GenBank database, and the accession number is EF428453．3．RT-PCR was conducted to amplify fragment of NPR1 gene by using primers derived from the conserved peptide motifs of cloned plant NPR1 from RNAs of sweet potato cultivar Qingnong no.2．The cDNA full-length of NPR1 was obtained by using RACE (rapid amplification of cDNA ends) method. The length of NPR1 was 2353 bp which included a 1761 bp-by complete open reading frame encoding NPR1 protein of 586 amino acids .The analysis of amino acid of NPR1 from sweet potato showed sweet potato NPR1 proteins included two domains of the POP/BTB and ANK domain, which were the conserved domains of NPR1．The ailgement of NPR1 from sweet potato with the NPR1 proteins of Arabidopsis, tobacco, tomato, rice, etc. showed that amino acids crucial for the NPR1 function, such as nprl-l(H), nprl-2(C), niml-4(R), are conserved. Sourth Blotting results showed the NPR1-related sequences were presented as 1-2 members in sweet potato genome. Semi-quantitative RT-PCR analysis showed the sweet potato NPR1 appear to be induced by salic acid (SA).The gene has been submitted to the GenBank database, and the accession number is EF190039．4．The plant expression vectors of SPR1 and NPR1 were constructed, it will be helpful to study their function. |
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