本文關(guān)鍵詞：草莓FaARF4基因的克隆鑒定及功能分析　出處：《沈陽農(nóng)業(yè)大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 草莓 ARF4 快速PCR定點突變 基因功能

【摘要】：ARF4(auxin response factor 4)是植物響應(yīng)生長素的轉(zhuǎn)錄因子,在植物生長素信號的轉(zhuǎn)導(dǎo)途徑中起著非常重要的作用。僅在模式植物中,ARF4的作用得到了較為清晰的研究,目前尚不清楚其在草莓中的作用。本試驗從'艷麗'草莓中克隆出ARF4基因編碼區(qū)序列,命名為FaARF4,構(gòu)建了點突變FaARF4基因編碼區(qū)(簡稱為FamARF4)的過表達(dá)載體,利用農(nóng)桿菌介導(dǎo)的轉(zhuǎn)化方法獲得了轉(zhuǎn)基因草莓和擬南芥的植株,為揭示草莓FaARF4基因的功能和作用機(jī)制打下基礎(chǔ)。本試驗的主要結(jié)果如下:1.FaARF4基因DNA全長5628 bp,FaARF4基因mRNA全長3236 bp,FaARF4基因編碼區(qū)序列序列全長2403 bp。利用擴(kuò)增'艷麗'草莓FaARF4基因編碼區(qū)序列的引物,在草莓栽培品種'全明星'、'甜查理'、'櫪乙女'、'豐香'和'紅顏'中都獲得了ARF 基因編碼區(qū)序列,發(fā)現(xiàn)它們之間氨基酸序列相似度為99.47%。2.利用探針法的qRT-PCR檢測了'艷麗'草莓不同器官中FaARF4的表達(dá)量,發(fā)現(xiàn)在幼葉、萼片、雌蕊中FaARF4的表達(dá)量較高。同時,本試驗還檢測了在草莓果實發(fā)育過程中FaARF4的表達(dá)量,發(fā)現(xiàn)在果實發(fā)育過程中FaARF4的表達(dá)呈下降的趨勢。3.運用快速PCR定點突變技術(shù)對FaARF4基因編碼區(qū)序列進(jìn)行改造,獲得了tasiRNA3的靶位點消除而其所翻譯的氨基酸序列不發(fā)生改變的點突變FaARF4基因編碼區(qū)序列。4.以植物過表達(dá)載體pRI 101 AN為基礎(chǔ)載體,利用Nde I/Sal I限制性核酸內(nèi)切酶,將草莓FamARF4結(jié)合到pRI 101 AN載體上,獲得過表達(dá)草莓FamARF4基因的植物表達(dá)載體,命名為pRI1 01-FamARF4。5.利用農(nóng)桿菌介導(dǎo)的葉盤轉(zhuǎn)化法,將'艷麗'草莓FamARF4基因?qū)氲缴植葺?Ruegen'中,獲得4株轉(zhuǎn)基因植株。利用農(nóng)桿菌介導(dǎo)的浸花轉(zhuǎn)化法,將'艷麗'草莓FamARF4基因?qū)氲綌M南芥中,獲得4株轉(zhuǎn)基因植株。6.通過觀察發(fā)現(xiàn),擬南芥和草莓轉(zhuǎn)基因植株都出現(xiàn)早開花的現(xiàn)象。另外,在擬南芥轉(zhuǎn)基因植株中,早期1-4朵花的敗育是由于雄蕊發(fā)育缺陷導(dǎo)致的;在草莓轉(zhuǎn)基因植株中,早期的果實上產(chǎn)生少量的種子,這種現(xiàn)象的原因有待進(jìn)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.
【學(xué)位授予單位】：沈陽農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S668.4

【參考文獻(xiàn)】

相關(guān)期刊論文 前9條

1 賈海鋒;趙密珍;王慶蓮;房經(jīng)貴;趙鵬程;劉眾杰;張成;糾松濤;;生長素和脫落酸在草莓果實發(fā)育過程中的作用[J];江蘇農(nóng)業(yè)科學(xué);2016年11期

2 周鶴瑩;張瑋;張卿;曹慶芹;沈元月;秦嶺;邢宇;;森林草莓‘Hawaii4’高效遺傳轉(zhuǎn)化系統(tǒng)的建立[J];北京農(nóng)學(xué)院學(xué)報;2015年01期

3 馮媛媛;李穎楠;侯佩;張景榮;汪松虎;劉永勝;;番茄ARF4基因果實特異RNAi載體的構(gòu)建及遺傳轉(zhuǎn)化[J];應(yīng)用與環(huán)境生物學(xué)報;2012年02期

4 王玉華;賈敬芬;;‘早紅’草莓高效遺傳轉(zhuǎn)化受體系統(tǒng)的建立[J];基因組學(xué)與應(yīng)用生物學(xué);2009年05期

5 吳霞,王嬌娟,朱禎,上官小霞,張林水,李波,杜春芳,李燕娥;轉(zhuǎn)Bt-CpTI-GNA基因棉花的研究(英文)[J];棉花學(xué)報;2005年06期

6 張紅梅,王俊麗;“全明星”草莓葉片遺傳轉(zhuǎn)化體系的建立[J];生物技術(shù);2005年03期

7 王俊麗,葛會波,彭士琪,張紅梅,續(xù)九如;草莓外源LEA_3基因的導(dǎo)入[J];園藝學(xué)報;2003年03期

8 張慧軍,石躍進(jìn),朱永紅,岳建雄,楊懷義;NPTII標(biāo)記轉(zhuǎn)基因棉花再生株的延期篩選[J];中國棉花;2002年05期

9 張志宏,吳祿平;草莓主栽品種Tudla遺傳轉(zhuǎn)化體系的建立[J];農(nóng)業(yè)生物技術(shù)學(xué)報;1998年02期

，

本文編號：1367712