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齿肋赤藓AP2/ERF转录因子家族基因Scsoloist的克隆与功能分析
李士敏
学位类型硕士
导师张道远
2016
学位授予单位中国科学院大学
学位授予地点北京
学位专业生物工程
关键词Ap2/erf转录因子 Scsoloist基因 齿肋赤藓 胁迫抗性
摘要植物为了适应环境,进化出了一系列的抗逆机制,而转录因子在其中起到非常重要的作用。AP2/ERF作为植物特有的转录因子参与了植物的抗胁迫信号途径,引起了众多的关注。但是对AP2/ERF转录因子的研究主要集中在DREB,ERF,RAV以及AP2亚家族,而对Soloist亚族报道极少。仅两篇文献报道Soloist亚族基因不仅参与了非生物胁迫还参与了生物胁迫的调控。为了更深入的了解Soloist亚族基因的功能,本研究克隆得到了极端耐干藓类植物齿肋赤藓(Syntrichia caninervis)的Soloist亚族基因Scsoloist及其上游启动子,并进行了生物信息学分析,同时对Scsoloist基因进行了表达模式、亚细胞定位、自激活活性分析,以及酵母和拟南芥的抗性分析。主要结果如下: 1、Scsoloist基因及其启动子的克隆与生物信息学分析:Scsoloist基因gDNA全长为955 bp,cDNA全长为561 bp,具有6个外显子和5个内含子;具有典型的AP2结构域;N端具有核定位信号肽,且定位于细胞核中;只与小立碗藓的亲缘关系较近,而与其它物种都较远;克隆得到Scsoloist基因上游1676 bp的核苷酸序列,预测发现了与抗逆性相关的以及一些新的作用元件。 2、Scsoloist基因在不同处理的表达模式:在复水条件下,Scsoloist基因随着处理时间的延长,表达量增加;在干旱和冷胁迫下,Scsoloist基因表达量先升高后下降;在SA和盐胁迫下Scsoloist基因表达量波动较大;在ABA处理下,Scsoloist基因在24 h出现表达高峰,随后恢复到正常水平。 3、亚细胞定位及自激活活性分析:通过基因枪轰击洋葱表皮细胞的方法证明ScSOLOIST蛋白定位于细胞核。酵母体系的激活实验证明,无论是Scsoloist基因整个片段还是截短后的系列基因片段(P1-P5),都没有表现出自激活活性,表明Scsoloist基因不具有转录激活域。 4、Scsoloist基因抗性功能分析:转Scsoloist基因酵母在渗透,盐,冷以及热胁迫下的长势与对照酵母长势相当,无显著差异。转Scsoloist基因拟南芥与野生型相比不具有抗盐胁迫的能力,但具有明显的对棉花黄萎病病菌大丽轮枝菌的抗性。
其他摘要In order to adjust to environment, plant has developed a series of resistance stress mechanisms, wherein transcription factors play a vital regular role. AP2/ERF transcription factors only exist in plant kingdom and have attracted considerable attention, among which too much more paid to the DREB, ERF, RAV and AP2 subfamilies than the Soloist subfamily. So far, only two articles demonstrated that the Soloist members had anti-abiotic and anti-biotic ability. For understanding more about the function of Soloist, we cloned the Scsoloist gene and its promotor sequence from Sytrichia carninervis, an extreme desiccation tolerance moss, and we conducted the bioinformatic analysis. At the same time we studied Scsoloist expression pattern, protein location and self-activation activity and resistence by yeast and Arabidopsis thaliana system. The main conclusions include four aspects as follows: 1. The cloning and bioinformatic analysis of Scsoloist gene and its promotor: The cDNA sequence of Scsoloist is 561 bp and gDNA which includes six exons and five introns is 955 bp. Scsoloist has a classical secondary structure of the AP2 domain: three β folder and one α helix. Subcellular location prediction shows that ScSOLOIST has nuclear localization signal peptide at the N-terminus and locates in cell nucleus. Phylogenetic tree analysis shows Scsoloist is close to Physcomitrella patens, but relative far to other species. we cloned a 1676 bp upstream sequence of Scsoloist,and by prediction, we found some cis-acting elements relating to stress tolerance and unreported new elements. 2. The expression patterns of Scsoloist under different treatments: Under rehydration, Scsoloist expression was enhanced with the lasted treating time. The transcript level of Scsoloist rose at first and then declined under drought and cold stresses. The Scsoloist expression was distinct between SA and salt treatment. Scsoloist responded to ABA after 24 h treatment, then recovered to the normal level. 3. Subcellular location and Self-activation analysis: By the method of particle bombardment to onion epidermal cells, ScSOLOIST was proved locating in cell nucleus. The activation experiment by yeast system shows neither whole Scsoloist nor truncated fragments (P1-P5) had self-activation ability. This result indicates that Scsoloist lacks transcription activating domain. 4. Scsoloist resistance function analysis: The growth of transgenic yeast was similar to the control ones under osmotic, salt, cold, and heat stresses, and transgenic yeast did not show better resistance ability. Compared to wild types, transgenic Arabidopsis thaliana did not show better growth conditions, but demonstrate obvious resistance to Verticillium dahlia which causes the cotton verticillium wilt.
学科领域生物工程
语种中文
文献类型学位论文
条目标识符http://ir.xjlas.org/handle/365004/14695
专题研究系统_荒漠环境研究室
作者单位中科院新疆生态与地理研究所
推荐引用方式
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李士敏. 齿肋赤藓AP2/ERF转录因子家族基因Scsoloist的克隆与功能分析[D]. 北京. 中国科学院大学,2016.
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