本文選題：擬南芥 + RNAi文庫�。� 參考：《鄭州大學》2016年碩士論文

【摘要】：本研究對擬南芥RNAi突變體文庫進行高溫脅迫篩選,獲得擬南芥突變體陽性單株,PCR鑒定測序獲得目的基因片段,長度為206bp,并命名該基因為At234。通過擬南芥TAIR網(wǎng)站數(shù)據(jù)庫序列比對結果發(fā)現(xiàn),At234位于擬南芥第2號染色體上,無內(nèi)含子結構,基因全長380bp,5’-UTR長度為44bp,3’-UTR長度為102bp,其ORF為234bp,編碼77個氨基酸殘基,所翻譯蛋白質的分子量為8.944kDa,等電點為9.98。為了進一步驗證該基因的功能,我們分別設定了RNAi植株與植物病原菌互作、重金屬脅迫、干旱脅迫和高鹽脅迫等生物與非生物脅迫驗證實驗。具體研究結果如下:確定野生型擬南芥的極限耐受高溫溫度閾值,最終采用65℃高溫分別處理擬南芥野生型與RNAi文庫(250庫與350庫)突變體各30min,在兩個RNAi文庫中都篩選出了若干株擬南芥耐極限高溫的RNAi突變體,通過PCR克隆獲得目的基因,均為At234基因的突變株。繼而通過潮霉素篩選培養(yǎng),單株收種,獲得擬南芥RNAi突變體的純合子。采用本實驗室自建的植物病原菌互作瓶內(nèi)檢測體系分析at234植株與病原菌的互作反應。研究發(fā)現(xiàn),在組培條件下at234植株較野生型擬南芥對棉花大麗輪枝菌和大豆核盤菌均表現(xiàn)為明顯感病;對玉米灰葡萄孢菌和茄病鐮刀菌均表現(xiàn)為明顯抗病;而在和小麥赤霉孢菌的互作中at234植株和野生型擬南芥均為正常感病。在組培條件下,at234植株較野生型擬南芥表現(xiàn)出高抗CdCl2(100mg/L)和Pb(NO3)2(1g/L)等重金屬鹽;在添加甘露醇(5%)的固體培養(yǎng)基中,at234植株較野生型擬南芥表現(xiàn)出明顯的抗旱性;在含有12g/L NaCl的高鹽脅迫培養(yǎng)基中,at234植株根系發(fā)育正常,表現(xiàn)出較強的高鹽耐受性。綜上所述,At234是擬南芥參與生物脅迫與非生物脅迫反應中的一個關鍵基因,其具體作用機制還有待進一步的研究。
[Abstract]:In this study, the RNAi mutant library of Arabidopsis thaliana was screened under high temperature stress.The results of TAIR database alignment of Arabidopsis thaliana showed that At234 was located on chromosome 2 of Arabidopsis thaliana and had no intron structure.The molecular weight of the translated protein is 8.944 kDa and the isoelectric point is 9.98.In order to further verify the function of the gene, we set up biological and abiotic stress verification experiments such as RNAi plant and plant pathogen interaction, heavy metal stress, drought stress and high salt stress.The specific results are as follows: determine the threshold of extreme tolerance to high temperature in wild type Arabidopsis thaliana.The wild-type Arabidopsis wild-type mutant and the RNAi library (U250 library and 350 library) were treated at 65 鈩，

本文編號：1743117