乙烯响应因子家族基因MdERF98的表达载体构建、苹果细胞转化与功能初步鉴定

发布时间：2018-12-17 22:12

【摘要】：乙烯是一重要植物激素,而在苹果这一以果实发育为重要研究对象的植物中,它的地位和作用又尤为重要和特殊,因为它调控果实的成熟;同时,乙烯又是植物抗病抗逆过程中重要的信号分子。乙烯响应因子(Ethylene responsive factor,ERF)基因是植物中特有的转录因子家族,调控植物的生长发育、抗性以及对乙烯等植物抗性相关激素的应答。我们实验室的前期研究结果表明,AP2/ERF转录因子家族中一转录因子MdERF98的表达能够被苹果轮纹病菌侵染所特异性诱导表达(张芮,2015),而该基因的功能以及在抗病抗逆中的作用,仍属未知,这引起我们强烈的兴趣。因此,在本研究中,首先应用生物信息学的方法,对该基因所属的苹果ERF基因家族的282个成员进行了生物信息学与进化树聚类分析;其次,克隆构建了MdERF98二元表达载体,进行了苹果愈伤组织细胞转化,成功获得超表达MdERF98转基因细胞株,对该转基因在抗盐中的作用进行了研究。主要研究结果如下:1.生物信息学与进化树分析表明:MdERF98与苹果基因组中MDP0000286915、MDP0000187369、MDP0000880063、MDP0000155543、MDP0000133854具有较高同源性,在进化树中,聚为一类,该类转录因子家族基因功能未知。同时,与在抗病反应中具有重要作用的经典的AP2转录因子家族基因Pti4没有聚为一类,相似性较低,同源关系较远。2.从苹果愈伤组织细胞中提取RNA,以反转录得到的cDNA为模板,应用PCR技术扩增得到MdERF98目的基因,将目的基因片段链接至中间载体pHBT上,用NcoI和Pst I双酶切获得目的基因片段,将目的基因成功连接至二元表达载体pCB302上,然后将表达载体转入农杆菌感受态细胞中,进一步用含有目的基因的农杆菌感受态细胞侵染野生型苹果‘王林’愈伤组织,获得转化MdERF98基因的‘王林’愈伤组织。3.分别对超表达MdERF98的苹果愈伤组织(王林)与野生型愈伤组织(王林)进行盐胁迫处理,共设置0 mM、25 mM、50 mM、100 mM、150 mM和200 mM六个NaCl的浓度梯度。结果表明:随着NaCl浓度的升高,愈伤组织的细胞生长量显著下降,但是,在盐分胁迫下,超表达MdERF98的转基因王林细胞的生长量显著高于野生型,MdERF98超表达显著提高了对盐分胁迫的抗性。4.与以拟南芥为研究试材的研究结果不同,在苹果中超表达MdERF98,没有显著提高维生素C的含量。推测,在苹果细胞中,MdERF98可能不是通过提高维生素C的含量以提高细胞的抗氧化能力而提高对盐分胁迫的抗性。5.对转MdERF98基因的苹果‘王林’愈伤组织与野生型‘王林’愈伤组织进行细胞含水量测定,设置三组重复。研究结果表明,转MdERF98基因的苹果‘王林’愈伤组织细胞含水量大于野生型。以上研究结果,为理解MdERF98的基因功能提供了新的信息,为进一步深入研究该基因功能奠定了基础。
[Abstract]:Ethylene is an important plant hormone, and in apple, which takes fruit development as an important research object, its position and function is especially important and special, because it regulates fruit maturation. At the same time, ethylene is an important signal molecule in plant disease resistance and stress resistance. Ethylene response factor (Ethylene responsive factor,ERF) gene is a unique transcription factor family in plants, which regulates plant growth and development, resistance and response to ethylene and other plant resistance related hormones. Previous studies in our laboratory have shown that the expression of a transcription factor MdERF98 in the AP2/ERF transcription factor family can be specifically induced by the infection of Rhizoctonia apple (Zhang Li, 2015). The function of the gene and its role in disease resistance and stress resistance remain unknown, which has aroused strong interest. Therefore, in this study, bioinformatics and phylogenetic tree cluster analysis were carried out for 282 members of the apple ERF gene family. Secondly, the binary expression vector of MdERF98 was cloned and constructed and transformed into callus cells of apple. The overexpression of MdERF98 transgenic cell line was successfully obtained, and the role of the transgenic gene in salt tolerance was studied. The main results are as follows: 1. Bioinformatics and phylogenetic tree analysis showed that MdERF98 had high homology with MDP0000286915,MDP0000187369,MDP0000880063,MDP0000155543,MDP0000133854 in apple genome. At the same time, the Pti4 gene of the classical AP2 transcription factor family, which plays an important role in disease resistance response, is not clustered into a class, and the similarity is low, and the homology is far. 2. RNA, was extracted from apple callus cells and reverse transcription cDNA was used as template. The target gene of MdERF98 was amplified by PCR technique. The target gene fragment was linked to the intermediate vector pHBT. The target gene fragment was digested with NcoI and Pst I to obtain the target gene fragment. The target gene was successfully ligated to the binary expression vector pCB302. The expression vector was transferred into Agrobacterium tumefaciens receptive cells to infect wild-type apple 'Wanglin' callus with Agrobacterium tumefaciens receptive cells containing the target gene. The callus of 'Wang Lin' transformed MdERF98 gene was obtained. The apple callus (Wang Lin) and the wild type callus (Wang Lin) were treated with salt stress, respectively. Six NaCl concentrations of 0 mM,25 mM,50 mM,100 mM,150 mM and 200 mM were set up. The results showed that the cell growth of callus decreased significantly with the increase of NaCl concentration. However, under salt stress, the growth of transgenic Wang Lin cells with overexpression of MdERF98 was significantly higher than that of wild type. Overexpression of MdERF98 significantly increased the resistance to salt stress. 4. Compared with Arabidopsis thaliana, overexpression of MdERF98, in apple did not significantly increase the content of vitamin C. It is speculated that in apple cells, MdERF98 may not increase the resistance to salt stress by increasing the content of vitamin C to increase the antioxidant ability of the cells. The water content of the callus of 'Wang Lin' and wild type 'Wanglin' were determined and repeated in three groups. The results showed that the water content of the callus of apple 'Wanglin' transgenic with MdERF98 gene was higher than that of wild type. These results provide new information for understanding the gene function of MdERF98 and lay a foundation for further study of the gene function.
【学位授予单位】：山东农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S661.1;Q943.2

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