Uroporphyrinogen III Synthase Gene Expression And Molecular Characteristic Analysis In Wheat Aibian1at The Condition Of Low Temperature

Wheat albinism line is a natural leaf color mutant from the parent dwarf wheat Aibian1,it is sensitive to low temperature. During winter and spring, leaves will be inducted to white,and when temperature rise up, the leaves will be back to green again. Wheat albinism line is astable genetic material. At present, from physiology, biochemistry, genetics and cytology, a lotof work have been done and got many important results. Based on previous research resultsthat the reason of albinism is frustration of enzymatic reaction which is betweenporphobilinogen(PBG) and urogen Ⅲ(Uro Ⅲ). So we picked these two enzymes whichparticipate in enzymatic reaction and upstream and downstream enzymes, they areporphobilinogen synthase(PBGS), hydroxymethylbilane synthase(PBGD), uroporphyrinigenⅢ synthase(UROS), uroporphyrinigen Ⅲ decarboxylase(UROD). The research used thealigment of four cDNAs, real-time PCR, transient expression of callus, methylation semi-realtime PCR to detect the difference of albinism line and aibian1, the results showed UROS maybe the major reason of albinism. The major results were:1. We cloned the cDNA sequence in FA85and Aibian1. The results of aligment showedthat the cDNA of PBGS and PBGD in FA85were similar to Aibian1, and no significantdifference. But UROS and UROD existed in many single base mutation, insertion anddeletion which contained termination codons, it can result in translation stopped early, secondarystructures were changed obviously.2. Using RACE (Rapid-amplification of cDNA ends) technology, we successfully got the5’end and the3’ end of UROS. The analysis results shows that the transcription initiation siteis located in the translation initiation codon ATG upstream about61bps; mRNA3’endsequence can form a hairpin structure, accord with typical termination sequence.3. Make use of LA PCR, we successfully got3sets of genomic sequences of UROS. Combined in silico cloning and Chinese Spring (CS) nulli-tetrasomics lines, we located3sets of genomic sequence3331,3314and3306bp to4A,4B and4D. Aligment4cDNAs with3gDNAs, there are2cDNA corresponding to D genomic sequence, so UROS-4D existed inthe progress of alternative splicing4. Make use of Real-Time PCR, we detected the expression of4cDNAs at the conditionof low temperature. UROS-4A kept low expression at both FA85and Aibian1, and noobvious relation to the time of low temperature. UROS-4B did not progress transcription, noexpression. UROS-4Da and UROS-4Db in FA85increased the level of expression followedby the increasing days, until the25thday the level of expression reduced sharply; UROS-4Daand UROS-4Db in Aibian expressed highly at normal temperature, but the level of expressionreduced slowly followed by the days, until the20thday the level rise up again. The result oftissue specific expression showed that UROS did not express in the germination of seeds,lowly expressed in root, and highly expressed in climax leaf and steam.5. Using in silico cloning, we get the promoter and terminater sequences of FA85andAibian1. After compassion, we knew two promoter sequence is highly similar, just a few ofbases difference. Using the software TRANSFAC v7.0we predicted that it contained a TATABox,3CAAT Box, and many putative transcriptional factor binding sites. The terminatersequence included a non-coding region beside the sequence which can stop transcription.6. We successfully built the transient expression vector PUROS::EGFP, using particlebombardment import it into mature embryos callus, observed by fluorescence microscopy.The results showed that GFP mainly concentrated in the cell membrane and the nucleus, andbrightness of PUROS::EGFP is weaker than P35S::EGFP. This means that the promoter have theactivity of transcription, and is lower than P35S::EGFP.7. Combine the enzyme McrBC and semi Real Time PCR, we get the change ofUROS-4D promoter at the condition of low temperature. The results indicated that the rate ofmethylation in Aibian1had no significant change, but in FA85at normal temperature the ratewas high, followed by the temperature reduced the rate fell down quickly, when the daysincreased the rate rise up again and keep stable.