利用蛋白質體學與反轉錄聚合酶連鎖反應研究有關 阿拉伯芥FIN219基因突變導致低溫抗性增加之原因

Abstract

Microarray資料的結果顯示，一種與抗凍相關的基因KIN2 ，於遠紅光下的基因表現量在WT與fin219突變株中具有顯著的差異。利用RT-PCR確認上述Microarray資料的結果是正確的。另外將WT與fin219及FIN219OE兩種轉殖株，於4℃下處理經3個月後，觀察低溫下的生長情形，結果發現fin219突變株生長情形優於FIN219OE轉殖株與WT。從PlantCare資料庫得知，FIN219基因啟動子上具有一個受低溫調控之C-repeat區域與一個受ABA調控區域ABA-Response-Element。FIN219 promer::GUS轉殖株經低溫處理與GUS活性染色，進一步確認FIN219啟動子是受低溫調控。 將WT、FIN219OE與fin219低溫下處理經2星期後，設計KIN2基因與FIN219基因專一性引子進行RT-PCR。藉此了解FIN219與KIN2基因在FIN219OE轉殖株與fin219突變株及WT中基因的表現。結果顯示，KIN2基因在fin219突變株比在FIN219OE與WT中具較高的表現量。 利用蛋白質體學研究工具，將三種不同品系：WT、fin219突變株、FIN219OE轉殖株，進行蛋白質二維膠體電泳。先了解三種品系的蛋白質表現，基本上有哪些不同的地方。結果發現14個蛋白質表現量有差異，其中有9-cis-epoxycarotenoid dioxygenase(A4)、acetyl-CoA carboxylase/ ligase(A8)putative AP2 domain containing protein RAP2.8(B2)、indole-3-glycerol-phosphate synthase(B6)ATEXLA2(Arabidopsis thaliana expansin-like A2)(A9)共五個蛋白質與低溫有關，分別在不同的FIN219基因表現下，具有表現量的差異藉此可以此解釋生理觀察結果中fin219突變株在低溫下的生長情形優於WT與FIN219OE的原因。

Microarray data indicated that KIN2 gene, which encodes antifreeze protein, showed different expression level among Wild type (WT) and fin219 mutant in far-red light. To confirm microarray data,total RNA were extracted WT、FIN219 overexpressor(FIN219OE) and fin219 treated with far-red light and KIN2 expression were examined by RT-PCR. As well these different plants were treated with 4℃ for 3 months to test their tolerance to cold stress. Result indicated that fin219 mutant was healthier than FIN219OE and WT. To future understand the conditions expression levels of FIN219 gene and KIN2 gene under stress, a RT-PCR approach was used for this.The result revealed that the KIN2 expression was higher in fin219 mutant than WT and FIN219OE , which indicates that the fin219 mutant is more chilling tolerant. The promoter sequence of the FIN219 was searched for cis-element revealed to the cold stress and found that C-repeat element and ABRE were present. To verify cold response,the FIN219 promoter::GUS transgenic plants were treated with cold and underwent for GUS-staining. The result indicated that FIN219 was also induced by cold. Proteomics tools helped to discover that 9-ciscaroteinoid dioxygenase(A4)、acetyl-CoA carboxylase/ ligase(A8)、ATEXLA2 (ARABIDOPSIS THALIANA EXPANSIN-LIKE A2)(A9)、putative AP2 domain containing protein RAP2.8(B2)、indole-3-glycerol-phosphate synthase(B6) have different expression patterns under various expression levels of the FIN219 gene and were involved in cold acclimation. Take together these data indicated that the fin219 mutant is more resistant to cold stresses than WT and FIN219OE plants.