Subcellular Localization Of XA21 And Identification Of Key Localized Domain

Xa21, Bacterial blight disease resistance gene from Rice (Oryza sativa L.), encodes a receptor-like kinase, which has key study value in molecular biology and genetics and breeding, and subcellular localization of its expression product XA21 protein is a key question in whole study.XA21 mostly be made up of Serine-threonine kinase (STK),Transmembrane (TM), Leucine-rich repeat (LRR), Postential signal sequence(PSS) from C-end to N-end in turn. Presumedly, TM with/without PSS play a role predominating subcellular localization of XA21. In this study, fusion green fluorescent protein(GFP) tag method is used to localize XA21 and identify key protein domain controlled localization.Five DNA fragments were cloned from Xa21 gene and named Xa21 FL (full Xa21 cDNA*), Xa21 2115, Xa21 1971, Xa21 1911 and Xa21 381, their size is 3126bp, 2115bp, 1971bp, 1911bp and 381bp respectively. Their 5'-terminal sequences are consistent with Xa21 cDNA* but DNA sequences encoding potential localization domains are deleted gradually from 3'-terminus. Xa21 FL is splited two entrons flanked one intron by recombination PCR method. Attentively, all of Xa21 DNA clones cover c-Myc with adapter sequence whose size is 48bp, Xa21 FL is equivalent to original Xa21 cDNA add to c-Myc sequence, signed Xa21 cDNA*. In the counterparts of proteins be expressed, except XA21 full protein, other proteins' STK, TM, smaller part of LRR and majority of LRR were deleted gradually from C-end. Construct 5 expression vectors which have target Xa21 clone fused to 5'-end of gene encoded GFP^S65T whose 65th amino acid replaces Serine with threonine, and establish check(CK) vector, pGFP^S65T embedded sequence encoding GFP^S65T. Subsequently, transform all vectors to onion epidermal cell via particle-mediated bombardment and incubate in MS culture medium for 24⁴8 hours, fusion protein with GFP^S65T were expressed in vitro and localization were analyzed under the assembling ternary form images by mean of confocal microscope. Due to these treats draw several conclusion as following:1. Obtained Xa21 FL clone which is equivalent to Xa21 cDNA* utilizing recombination PCR method.2. Established 5 expression plasmid vectors fused target Xa21 clone with gene encoded GFP8657, namely pXa21 381-GFPS65T,pXa21 1911-GFPS65T,pXa21 1971-GFPS65T,pXa21 2115-GFPS65randpXa21 FL-GFP55651.3. The analysis result derived from fluorescent image indicate that fluorescent distribution pattern of CK is nearly overall cellular compartment without preference, therefore GFPS65T was not proved impact fusion of targeted protein to localize to self-appointed compartment. The XA21-GFPS65T appear to localize to the plasma membrane where was focused fluorescence exclusively.4. In order to identify center domain controlled subcellular localization utilizing differential analysis method that difference fluorescent signal distribution patterns of fusion fluorescent protein(FP) with deletion protein compare with that of fusion FP with whole XA21 (XA21-GFPS65T). 4 deletion fusion proteins besides plasma membrane for nucleus and so forth. Consequently elementary result have been found to be required for the normal localization keeping integrality of XA21 protein, but could not merely depend on putative TM with/sans PSS.