Real-time Fluorescent Quantitative Analysis Of Wheat Ice Recrystallization Inhibition TaIRI Gene Of Shumai482under Cold Stress

Based on the homologous cloning, two gene sequences encoding TaIRI protein (Triticum aestivum ice recrystallization inhibition) were separated from common wheat Shumai482and China Spring. Genetic variations of TaIRI in molecular level were detected in the chilling stress. Meanwhile, alignment analysis and fluorescence quantitative expression analysis of the genetic sequence of TaIRI were carried out. On that basis, expression difference of TaIRI was researched between cold resistance wheat and non-cold resistance wheat, which could clarify the expression of TaIRI in different cold resistance wheat during different period. The following results were obtained.1. Shumai482and China Spring dealed with4℃were observed, as time goes on, China Spring died while Shumai482was alive. So, Shumai482was regarded as cold resistance wheat varieties relative to China Spring. The two wheat varieties were selected as the test subjects because they had great difference in cold resistance.2. The two putative TaIRI gene sequences from the two wheat varieties were859bp and865bp through literature reviewing and NCBI alignment. Through alignment analysis by DNAman, two coding regions have not intron and include insertion, deletion, transversion and transation. After blastn of NCBI, the sequences got from Shumai482and China spring were confirmed as TaIRI genes (Triticum aestivum ice recrystallization inhibition).3. Gene specific primers of Real-Time Quantitative RT-PCR were designed according to the two putative TaIRI gene sequences. After testing, the lasted specific primers were named as TaIRI-1,TaIRI-2,TaIRI-3and TaIRI-4. In addition, conventional Actin and newly developed Ta-at from laboratory were selected as housekeeping genes. All primers were tested and verified, so that they can be used for quantitative fluorescence PCR test.4. Total RNA of Shumai482were extracted in common condition (Ohr and25℃).Then the two wheat varieties were disposed at4℃and12hr,24hr,48hr,96hr and144hr, respectively. And total RNA of root and leaf from five periods were extracted and used for reversed transcription. The results showed that, within12h following cold stress, the TaIRI gene expressive level has significant difference. The expression of Root and leaf of Shumai482were significantly higher than China Spring. Expression pattern rised and then falled and48hr or96hr was peak. Among others, expression of TaIRI-4gene did not show in Ohr, which was considered that TaIRI-4gene did not express within cold stress. The experimental result was similar to the conclusion of Tremblay K and so on. But, the difference was that the peak of expression level in this experiment was the period of48hr or96hr, nevertheless, the result of Tremblay K showed that the peak was6d or36d.