乙烯響應(yīng)因子家族基因MdERF98的表達載體構(gòu)建、蘋果細胞轉(zhuǎn)化與功能初步鑒定
發(fā)布時間：2018-12-17 22:12
【摘要】：乙烯是一重要植物激素,而在蘋果這一以果實發(fā)育為重要研究對象的植物中,它的地位和作用又尤為重要和特殊,因為它調(diào)控果實的成熟;同時,乙烯又是植物抗病抗逆過程中重要的信號分子。乙烯響應(yīng)因子(Ethylene responsive factor,ERF)基因是植物中特有的轉(zhuǎn)錄因子家族,調(diào)控植物的生長發(fā)育、抗性以及對乙烯等植物抗性相關(guān)激素的應(yīng)答。我們實驗室的前期研究結(jié)果表明,AP2/ERF轉(zhuǎn)錄因子家族中一轉(zhuǎn)錄因子MdERF98的表達能夠被蘋果輪紋病菌侵染所特異性誘導(dǎo)表達(張芮,2015),而該基因的功能以及在抗病抗逆中的作用,仍屬未知,這引起我們強烈的興趣。因此,在本研究中,首先應(yīng)用生物信息學(xué)的方法,對該基因所屬的蘋果ERF基因家族的282個成員進行了生物信息學(xué)與進化樹聚類分析;其次,克隆構(gòu)建了MdERF98二元表達載體,進行了蘋果愈傷組織細胞轉(zhuǎn)化,成功獲得超表達MdERF98轉(zhuǎn)基因細胞株,對該轉(zhuǎn)基因在抗鹽中的作用進行了研究。主要研究結(jié)果如下:1.生物信息學(xué)與進化樹分析表明:MdERF98與蘋果基因組中MDP0000286915、MDP0000187369、MDP0000880063、MDP0000155543、MDP0000133854具有較高同源性,在進化樹中,聚為一類,該類轉(zhuǎn)錄因子家族基因功能未知。同時,與在抗病反應(yīng)中具有重要作用的經(jīng)典的AP2轉(zhuǎn)錄因子家族基因Pti4沒有聚為一類,相似性較低,同源關(guān)系較遠。2.從蘋果愈傷組織細胞中提取RNA,以反轉(zhuǎn)錄得到的cDNA為模板,應(yīng)用PCR技術(shù)擴增得到MdERF98目的基因,將目的基因片段鏈接至中間載體pHBT上,用NcoI和Pst I雙酶切獲得目的基因片段,將目的基因成功連接至二元表達載體pCB302上,然后將表達載體轉(zhuǎn)入農(nóng)桿菌感受態(tài)細胞中,進一步用含有目的基因的農(nóng)桿菌感受態(tài)細胞侵染野生型蘋果‘王林’愈傷組織,獲得轉(zhuǎn)化MdERF98基因的‘王林’愈傷組織。3.分別對超表達MdERF98的蘋果愈傷組織(王林)與野生型愈傷組織(王林)進行鹽脅迫處理,共設(shè)置0 mM、25 mM、50 mM、100 mM、150 mM和200 mM六個NaCl的濃度梯度。結(jié)果表明:隨著NaCl濃度的升高,愈傷組織的細胞生長量顯著下降,但是,在鹽分脅迫下,超表達MdERF98的轉(zhuǎn)基因王林細胞的生長量顯著高于野生型,MdERF98超表達顯著提高了對鹽分脅迫的抗性。4.與以擬南芥為研究試材的研究結(jié)果不同,在蘋果中超表達MdERF98,沒有顯著提高維生素C的含量。推測,在蘋果細胞中,MdERF98可能不是通過提高維生素C的含量以提高細胞的抗氧化能力而提高對鹽分脅迫的抗性。5.對轉(zhuǎn)MdERF98基因的蘋果‘王林’愈傷組織與野生型‘王林’愈傷組織進行細胞含水量測定,設(shè)置三組重復(fù)。研究結(jié)果表明,轉(zhuǎn)MdERF98基因的蘋果‘王林’愈傷組織細胞含水量大于野生型。以上研究結(jié)果,為理解MdERF98的基因功能提供了新的信息,為進一步深入研究該基因功能奠定了基礎(chǔ)。
[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.
【學(xué)位授予單位】：山東農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：S661.1;Q943.2
本文編號：2384866
[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.
【學(xué)位授予單位】：山東農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：S661.1;Q943.2
【參考文獻】
相關(guān)期刊論文 前10條
1 季愛加;羅紅梅;徐志超;張鑫;宋經(jīng)元;陳士林;;藥用植物轉(zhuǎn)錄因子AP2/ERF研究與展望[J];科學(xué)通報;2015年14期
2 張計育;王慶菊;郭忠仁;;植物AP2/ERF類轉(zhuǎn)錄因子研究進展[J];遺傳;2012年07期
3 ;Functions and Application of the AP2/ERF Transcription Factor Family in Crop Improvement[J];Journal of Integrative Plant Biology;2011年07期
4 ;Salicylic Acid and its Function in Plant Immunity[J];Journal of Integrative Plant Biology;2011年06期
5 郭艷;楊海玲;;植物組織培養(yǎng)中的褐化現(xiàn)象及解決途徑[J];山西農(nóng)業(yè)科學(xué);2009年07期
6 葛春輝;計巧靈;王雪華;賈紅麗;朱國麗;張丕鴻;;亞麻耐鹽性愈傷組織的生理生化特性[J];植物研究;2008年05期
7 陳霞;羅世巧;段翠芳;曾日中;;高等植物轉(zhuǎn)錄因子研究進展[J];安徽農(nóng)學(xué)通報;2008年09期
8 李曉星;蔣海雄;黃青云;夏令;張紅心;張大兵;陳亮;;水稻bHLH基因家族成員表達模式分析[J];廈門大學(xué)學(xué)報(自然科學(xué)版);2006年S1期
9 羅賽男,楊國順,石雪暉,盧向陽,徐萍;轉(zhuǎn)錄因子在植物抗逆性上的應(yīng)用研究[J];湖南農(nóng)業(yè)大學(xué)學(xué)報(自然科學(xué)版);2005年02期
10 李鳳華,湯紹虎,孫一銘,孫敏;降低何首烏愈傷組織褐化研究[J];西南師范大學(xué)學(xué)報(自然科學(xué)版);2005年02期
相關(guān)博士學(xué)位論文 前1條
1 張芮;蘋果MdWRKY33基因在輪紋病抗性形成中的作用機制研究[D];山東農(nóng)業(yè)大學(xué);2015年
相關(guān)碩士學(xué)位論文 前4條
1 王榮凱;蘋果MdCIPK6的基因克隆及其在逆境脅迫響應(yīng)中的作用[D];山東農(nóng)業(yè)大學(xué);2011年
2 尹蓉;蘋果屬植物幼苗對鹽脅迫的耐性評價及生理響應(yīng)[D];西北農(nóng)林科技大學(xué);2010年
3 張燕飛;雜交新美柳扦插苗NaCl脅迫及組織培養(yǎng)研究[D];南京林業(yè)大學(xué);2007年
4 李增裕;蘋果砧木耐鹽變異細胞系篩選和鑒定指標(biāo)的研究[D];河北農(nóng)業(yè)大學(xué);2003年
，本文編號：2384866
本文鏈接：http://sikaile.net/kejilunwen/jiyingongcheng/2384866.html
最近更新
教材專著
