|
|
|
Abstract An unique expressed sequence tag（EST）, which had the high similarity to the sequence of Arabidopsis thaliana LRR Cf-5 disease resistance protein-like, was found from an SSH cDNA library constructed using mRNA isolated from leaves of Chinese cabbage (Brassica campestris ssp. pekinensis) after Ecc (Erwinia carotovora subsp.carotovora) infection. RACE was used to extend the 5’and 3’unknown sequence of this EST. The cloned cDNA, named BcLRR, was 1286 bp including a complete open reading frame (ORF) and a complete 3’UTR with a putative poly (A) signal (GenBank Accession No.EU424347). The deduced polypeptide contained 327 amino acids and consisted of transmembrance domain on the N sequences and 8 extra-cytoplasmic LRR motifs, 7 of which had canonical LXXLXLXXN repeat units and one had non-canonical LXXLXXXXN unit. Amino acid sequence alignment analysis revealed that BcLRR displayed high homology of 96.6% with Arabidopsis Cf-5 and of 82.3％ with upland cotton LRR disease resistance protein-like GhLRR; The majority of their differences are confined to their N terminal signal peptide regions. Phylogenetic analysis further demonstrated that these three functionally unknown proteins were significantly distinct from other known resistance extra-cytoplasmic LRR proteins (Cf-2, Cf-4, Cf-5, and Cf-9 from tomato, Hs1pro-1 from sugarbeet and Xa21 from rice) and belonged to a new type of extra-cytoplasmic LRR protein. The complete ORF sequence of BcLRR was amplified and cloned into modified vector pGreen0029 under CaMV35S promoter, forming plant expressed vector pGreen0029-BcLRR, which was then transformed into Arabidopsis thaliana Col-0 via Agrobacterium GV3101/Soup. The results of PCR, Southern blotting and RT-PCR showed that BcLRR had been integrated into the Arabidopsis genome and expressed in the transgenic plants. BcLRR protein enhanced the resistance to Ecc BC1 in the transgentic Arabidopsisp plant.
|
|
Received: 08 September 2009
|
|
|
|
|
|
