花生促丝裂原蛋白活化激酶基因的克隆与表达分析

发布时间：2018-05-27 10:06

  本文选题：MAPK胀基因 + 花生　； 参考：《新疆农业大学》2016年硕士论文

【摘要】：随着农业的进步和发展,花生育种越来越受重视。在植物生长发育中经常遭遇外界伤害,虽然不能通过移动来逃避伤害,但它能快速感知外界信号来主动适应环境。以往的研究表明,植物应激抗逆性反应时,促丝裂原蛋白活化激酶(MAPK)能发挥重要作用。本研究以花育19为实验材料和拟南芥MAPK蛋白序列AtMAPKl(GenBank:NP_181907.1)为参考序列,利用同源比对法和RT_PCR, RACE-PCR方法从花生中克隆到一个新的MAPK基因并对其进行了序列比对和表达特性分析。具体结果如下：(1)利用花生研究中心构建的花生cDNA文库和拟南芥MAPK蛋白序列AtMAPK1 (GenBank:NP_181907.1)为参考序列,使用EST测序和RACE克隆等方法,克隆得到了一个MAPK基因。其1492bp是全长cDNA序列,含有1191bp的开放阅读框架(ORF),能编码397个氨基酸的蛋白质序列。(2)4个物种中MAPK基因的氨基酸序列比对,发现该基因含有典型的Ser/Thr保守基序,与拟南芥、大豆、本生烟的同源性分别为86%、94%和90%。结果发现与拟南芥MAPK6的同源和进化关系密切可以归属于MAPK家族的A组。将其命名为AhMAPK6a,并在GenBank上注册,注册号为KP329549。(3)采用荧光定量方法对AhMAPK6a进行组织特异性表达分析,发现该基因在叶片和根系中的表达量最高,花,子叶和茎中表达量低,具有明显的组织表达特异性。(4)采用荧光定量方法对花生AhMAPK6a在ABA,PEG,JA,SA处理下,进行表达特异性分析,该基因表达量发生明显变化。在ABA处理的花生叶和根中,AhMAPK6a基因表达量先短暂减少后增加,之后几小时该基因的表达量并没有明显变化。在15%PEG处理叶片中,基因表达量明显下降,但在根中的表达量先短暂减少后增加,之后几个小时内基因表达量没有变化。在JA处理中表现为显著正增长的基因表达水平;在SA处理中表现为负增长的基因表达水平。(5)使用农杆菌EHA105转化构建好的正义基因表达载体pCAMBIA1301-AhMAPK6a,使用含有目的基因的农杆菌浸润拟南芥未开放的花蕾。获得侵染过的拟南芥种子后将其灭菌后置于Kan(20mg/L)的1/2MS培养基平板上,4℃黑暗中放置3-4天,培养室黑暗培养一周左右,选择长的健壮拟南芥幼苗进行PCR鉴定得到了5株阳性植株。本文为研究花生的抗逆分子机理及抗逆基因工程提供科学依据,为获得花生抗逆品种提供候选基因。
[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，

本文编号：1941569