草莓FaARF4基因的克隆鉴定及功能分析

发布时间：2018-01-02 04:30

  本文关键词：草莓FaARF4基因的克隆鉴定及功能分析　出处：《沈阳农业大学》2017年硕士论文　论文类型：学位论文

  更多相关文章： 草莓 ARF4 快速PCR定点突变 基因功能

【摘要】：ARF4(auxin response factor 4)是植物响应生长素的转录因子,在植物生长素信号的转导途径中起着非常重要的作用。仅在模式植物中,ARF4的作用得到了较为清晰的研究,目前尚不清楚其在草莓中的作用。本试验从'艳丽'草莓中克隆出ARF4基因编码区序列,命名为FaARF4,构建了点突变FaARF4基因编码区(简称为FamARF4)的过表达载体,利用农杆菌介导的转化方法获得了转基因草莓和拟南芥的植株,为揭示草莓FaARF4基因的功能和作用机制打下基础。本试验的主要结果如下:1.FaARF4基因DNA全长5628 bp,FaARF4基因mRNA全长3236 bp,FaARF4基因编码区序列序列全长2403 bp。利用扩增'艳丽'草莓FaARF4基因编码区序列的引物,在草莓栽培品种'全明星'、'甜查理'、'枥乙女'、'丰香'和'红颜'中都获得了ARF 基因编码区序列,发现它们之间氨基酸序列相似度为99.47%。2.利用探针法的qRT-PCR检测了'艳丽'草莓不同器官中FaARF4的表达量,发现在幼叶、萼片、雌蕊中FaARF4的表达量较高。同时,本试验还检测了在草莓果实发育过程中FaARF4的表达量,发现在果实发育过程中FaARF4的表达呈下降的趋势。3.运用快速PCR定点突变技术对FaARF4基因编码区序列进行改造,获得了tasiRNA3的靶位点消除而其所翻译的氨基酸序列不发生改变的点突变FaARF4基因编码区序列。4.以植物过表达载体pRI 101 AN为基础载体,利用Nde I/Sal I限制性核酸内切酶,将草莓FamARF4结合到pRI 101 AN载体上,获得过表达草莓FamARF4基因的植物表达载体,命名为pRI1 01-FamARF4。5.利用农杆菌介导的叶盘转化法,将'艳丽'草莓FamARF4基因导入到森林草莓'Ruegen'中,获得4株转基因植株。利用农杆菌介导的浸花转化法,将'艳丽'草莓FamARF4基因导入到拟南芥中,获得4株转基因植株。6.通过观察发现,拟南芥和草莓转基因植株都出现早开花的现象。另外,在拟南芥转基因植株中,早期1-4朵花的败育是由于雄蕊发育缺陷导致的;在草莓转基因植株中,早期的果实上产生少量的种子,这种现象的原因有待进一步研究。
[Abstract]:ARF4(auxin response factor 4 is the transcription factor of plant response to auxin. It plays a very important role in the transduction pathway of plant auxin signal. The role of ARF4 in model plants has been clearly studied. At present, the role of ARF4 gene in strawberry is not clear. In this study, the coding region of ARF4 gene was cloned from 'showy' strawberry and named FaARF4. The overexpression vector of point mutant FaARF4 gene coding region (FamARF4) was constructed and transgenic strawberry and Arabidopsis plants were obtained by Agrobacterium tumefaciens-mediated transformation. In order to reveal the function and mechanism of strawberry FaARF4 gene, the main results of this experiment are as follows: 1. The total length of DNA of FaARF4 gene is 5 628 BP. The mRNA of FaARF4 gene was 3236bp in length. The sequence of FaARF4 gene coding region was 2403 BP. The primers amplified the coding region of 'Yanli' strawberry FaARF4 gene were used in strawberry cultivar 'All-Star'. The coding region of ARF gene was obtained in Carpinus pinpinus' Fengxiang 'and' Hongyan'. It was found that the similarity of amino acid sequence between them was 99.47.2.The expression of FaARF4 in different organs of 'showy' strawberry was detected by qRT-PCR with probe method, and the expression of FaARF4 was found in young leaves. The expression of FaARF4 was higher in sepals and pistil. At the same time, the expression of FaARF4 was detected during the development of strawberry fruit. It was found that the expression of FaARF4 decreased during fruit development. Rapid PCR site-directed mutation technique was used to modify the coding region of FaARF4 gene. The point mutation FaARF4 gene coding region. 4. The target elimination of tasiRNA3 was obtained, but the amino acid sequence did not change. 4. The plant overexpression vector pRI 101 was used as a plant overexpression vector. An is the base carrier. Strawberry FamARF4 was conjugated to pRI 101an vector by Nde I / Sal I restriction endonuclease. A plant expression vector named pRI1 01-FamARF4.5was obtained which expressed the FamARF4 gene of strawberry. Agrobacterium tumefaciens mediated leaf disc transformation was used. The FamARF4 gene of 'Yanli' strawberry was introduced into Ruegen 'and 4 transgenic plants were obtained. Agrobacterium tumefaciens mediated flower transformation was used. The FamARF4 gene of 'Yanli' strawberry was introduced into Arabidopsis thaliana and 4 transgenic plants. 6. Through observation, it was found that both Arabidopsis and strawberry transgenic plants appeared the phenomenon of early flowering. In Arabidopsis thaliana transgenic plants, the early abortion of 1-4 flowers was caused by stamen defect. In strawberry transgenic plants, a small number of seeds were produced on the early fruits, and the causes of this phenomenon need to be further studied.
【学位授予单位】：沈阳农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S668.4

【参考文献】

相关期刊论文 前9条

1 贾海锋;赵密珍;王庆莲;房经贵;赵鹏程;刘众杰;张成;纠松涛;;生长素和脱落酸在草莓果实发育过程中的作用[J];江苏农业科学;2016年11期

2 周鹤莹;张玮;张卿;曹庆芹;沈元月;秦岭;邢宇;;森林草莓‘Hawaii4’高效遗传转化系统的建立[J];北京农学院学报;2015年01期

3 冯媛媛;李颖楠;侯佩;张景荣;汪松虎;刘永胜;;番茄ARF4基因果实特异RNAi载体的构建及遗传转化[J];应用与环境生物学报;2012年02期

4 王玉华;贾敬芬;;‘早红’草莓高效遗传转化受体系统的建立[J];基因组学与应用生物学;2009年05期

5 吴霞,王娇娟,朱祯,上官小霞,张林水,李波,杜春芳,李燕娥;转Bt-CpTI-GNA基因棉花的研究(英文)[J];棉花学报;2005年06期

6 张红梅,王俊丽;“全明星”草莓叶片遗传转化体系的建立[J];生物技术;2005年03期

7 王俊丽,葛会波,彭士琪,张红梅,续九如;草莓外源LEA_3基因的导入[J];园艺学报;2003年03期

8 张慧军,石跃进,朱永红,岳建雄,杨怀义;NPTII标记转基因棉花再生株的延期筛选[J];中国棉花;2002年05期

9 张志宏,吴禄平;草莓主栽品种Tudla遗传转化体系的建立[J];农业生物技术学报;1998年02期

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