本文選題：MAPK脹基因 + 花生��； 參考：《新疆農(nóng)業(yè)大學(xué)》2016年碩士論文

【摘要】：隨著農(nóng)業(yè)的進步和發(fā)展,花生育種越來越受重視。在植物生長發(fā)育中經(jīng)常遭遇外界傷害,雖然不能通過移動來逃避傷害,但它能快速感知外界信號來主動適應(yīng)環(huán)境。以往的研究表明,植物應(yīng)激抗逆性反應(yīng)時,促絲裂原蛋白活化激酶(MAPK)能發(fā)揮重要作用。本研究以花育19為實驗材料和擬南芥MAPK蛋白序列AtMAPKl(GenBank:NP_181907.1)為參考序列,利用同源比對法和RT_PCR, RACE-PCR方法從花生中克隆到一個新的MAPK基因并對其進行了序列比對和表達特性分析。具體結(jié)果如下：(1)利用花生研究中心構(gòu)建的花生cDNA文庫和擬南芥MAPK蛋白序列AtMAPK1 (GenBank:NP_181907.1)為參考序列,使用EST測序和RACE克隆等方法,克隆得到了一個MAPK基因。其1492bp是全長cDNA序列,含有1191bp的開放閱讀框架(ORF),能編碼397個氨基酸的蛋白質(zhì)序列。(2)4個物種中MAPK基因的氨基酸序列比對,發(fā)現(xiàn)該基因含有典型的Ser/Thr保守基序,與擬南芥、大豆、本生煙的同源性分別為86%、94%和90%。結(jié)果發(fā)現(xiàn)與擬南芥MAPK6的同源和進化關(guān)系密切可以歸屬于MAPK家族的A組。將其命名為AhMAPK6a,并在GenBank上注冊,注冊號為KP329549。(3)采用熒光定量方法對AhMAPK6a進行組織特異性表達分析,發(fā)現(xiàn)該基因在葉片和根系中的表達量最高,花,子葉和莖中表達量低,具有明顯的組織表達特異性。(4)采用熒光定量方法對花生AhMAPK6a在ABA,PEG,JA,SA處理下,進行表達特異性分析,該基因表達量發(fā)生明顯變化。在ABA處理的花生葉和根中,AhMAPK6a基因表達量先短暫減少后增加,之后幾小時該基因的表達量并沒有明顯變化。在15%PEG處理葉片中,基因表達量明顯下降,但在根中的表達量先短暫減少后增加,之后幾個小時內(nèi)基因表達量沒有變化。在JA處理中表現(xiàn)為顯著正增長的基因表達水平;在SA處理中表現(xiàn)為負增長的基因表達水平。(5)使用農(nóng)桿菌EHA105轉(zhuǎn)化構(gòu)建好的正義基因表達載體pCAMBIA1301-AhMAPK6a,使用含有目的基因的農(nóng)桿菌浸潤擬南芥未開放的花蕾。獲得侵染過的擬南芥種子后將其滅菌后置于Kan(20mg/L)的1/2MS培養(yǎng)基平板上,4℃黑暗中放置3-4天,培養(yǎng)室黑暗培養(yǎng)一周左右,選擇長的健壯擬南芥幼苗進行PCR鑒定得到了5株陽性植株。本文為研究花生的抗逆分子機理及抗逆基因工程提供科學(xué)依據(jù),為獲得花生抗逆品種提供候選基因。
[Abstract]:With the progress and development of agriculture, peanut breeding has been paid more and more attention. Plant growth and development often encounter external injuries, although it can not be moved to avoid injury, but it can quickly sense the external signals to adapt to the environment. Previous studies have shown that mitogen-activated kinase (MAPK) may play an important role in plant stress response. In this study, a new MAPK gene was cloned from peanut by using homology alignment method and RACE-PCR method, and a new MAPK gene was cloned from peanut by using homology alignment method and RACE-PCR method. A new MAPK gene was cloned from peanut and analyzed its sequence alignment and expression characteristics by using floral 19 and Arabidopsis MAPK protein sequence AtMAPKln GenBank: NP181907.1 as reference sequence. The specific results are as follows: (1) A MAPK gene was cloned by using the peanut cDNA library and Arabidopsis MAPK protein sequence AtMAPK1 GenBank: NPV 181907.1) constructed by peanut research center. EST sequencing and RACE cloning were used to clone a MAPK gene. Its 1492bp is a full-length cDNA sequence, an open reading frame containing 1191bp, which encodes a 397-amino acid protein sequence. The amino acid sequences of the MAPK gene in four species were compared. It was found that the MAPK gene contained a typical conserved Ser/Thr motif with Arabidopsis thaliana and soybean. The homology was 86% and 90%, respectively. The results showed that the homology and evolution of Arabidopsis MAPK6 were closely related to group A of MAPK family. It was named AhMAPK6a and registered on GenBank (registration number KP329549.3). The expression of AhMAPK6a was analyzed by fluorescence quantitative method. It was found that the expression of the gene was the highest in leaves and roots, and low in flowers, cotyledons and stems. The expression specificity of peanut AhMAPK6a was analyzed by fluorescence quantitative method. In peanut leaves and roots treated with ABA, the expression of AhMAPK6a gene decreased briefly and then increased, but did not change significantly after a few hours. In the leaves treated with 15%PEG, the gene expression decreased significantly, but the expression in the root decreased briefly and then increased, but the gene expression did not change within a few hours. The level of gene expression was significantly positive in JA treatment. The sense gene expression vector pCAMBIA1301-AhMAPK6awas constructed by EHA105 transformation of Agrobacterium tumefaciens. Agrobacterium tumefaciens containing the target gene was used to infiltrate the unopened flower buds of Arabidopsis thaliana. After obtaining infected Arabidopsis thaliana seeds and sterilizing them, they were placed on the plate of 1/2MS medium (20 mg / L) in darkness for 3-4 days at 4 鈩，

本文編號：1941569