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齿肋赤藓早期光诱导蛋白基因ELIP的克隆及功能分析
周雅
学位类型硕士
导师张道远
2015
学位授予单位中国科学院大学
学位授予地点北京
学位专业植物学
关键词齿肋赤藓 早期光诱导蛋白 光保护 表达模式 功能验证
摘要齿肋赤藓是一种极端耐旱藓类,能在失水98%以上条件下存活,一旦遇水,30s内迅速复苏,本研究从齿肋赤藓转录组数据库出发,发现复水过程中齿肋赤藓的色素结合蛋白全部上调表达,其中早期光诱导蛋白蛋白(Early light-induced protein,ELIPs)表达最早,它属于叶绿素结合蛋白(Chlorophyll a/b binding Proteins,CABs)超家族,是一类定位于叶绿体类囊体膜上的跨膜蛋白,在蓝藻、苔藓、高等植物中都有发现,其最早在黄化的豌豆幼苗转绿过程中被发现,因其受诱导的时间比其他光诱导蛋白都早,并在光合器官发育完整后迅速消失,具有瞬时积累的特性而得名。本研究对齿肋赤藓体内的ELIP基因进行了家族分析,进一步筛选出两条具有全长的ScELIP基因,分别对其进行了生物信息学分析、基因克隆、表达模式分析和功能验证,结果如下: 1. 本研究从齿肋赤藓体内共获得39条注释为早期光诱导蛋白的unigenes,经鉴定后获得27条ScELIP基因,其中6条含完整ORF,且具有chloro_a/b binding保守域。 2. 本研究筛选2条ScELIPs 基因进行生物信息学分析,二者ORF全长分别为711bp和624bp,分别编码236和207个氨基酸,具有Chloro_a/b binding功能域,定位于叶绿体类囊体膜,含有3个跨膜α螺旋。氨基酸序列比对分析得出ScELIP1与藻类、苔藓聚为一支,而ScELIP2与维管植物聚为一支,表现出ELIP基因从低等到高等植物的进化特征。 3. 本研究对两条ScELIPs基因进行实验克隆,并研究了其在各种非生物胁迫及不同光照条件下的表达模式:①ScELIP1对快干、慢干、NaCl、冷、热等非生物胁迫都响应,ScELIP2对快干胁迫不响应,二者对NaCl、冷胁迫的响应均快速而持久;②ScELIP1和ScELIP2对UVA、UVB胁迫均有相应,但ScELIP1对UVA的响应微弱且有瞬时性,而ScELIP2的响应明显且持久;蓝光诱导ScELIPs表达且促进红光的诱导作用,红光对蓝光的诱导有抑制性。ScELIP1和ScELIP2的表达量随着光照强度的升高不断提高。 4. 本研究利用转基因酵母来进行ScELIP功能验证。结果发现转基因酵母与对照组酵母在各种胁迫(20% PEG、5mol/L NaCl、-20℃、53℃、UV)下的生长情况基本一致,即ScELIP基因在酵母体内未表现出非生物胁迫抗性和光保护的功能。本研究进一步在模式植物拟南芥中验证ScELIP的功能。通过比较稳定遗传的转ScELIP2基因拟南芥与对照组在强光、UV、红光、蓝光、干、盐、冷、热等多种胁迫下的生长情况及胁迫相关的生理指标测量与比较,得出ScELIP2基因具有非生物胁迫抗性和光保护作用。
其他摘要Syntrichia caninervis is an extremly drought-resistant moss which can survive in losing 98% body water. Once given water, it can recover rapidly in 30s. Based on the transcription data of S. caninervis, we found that the chorophyll a/b binding proteins are all up regulated. Among these proteins, Early light induced proteins expressed early than others. They belong to the chlorophyll a/b-binding protein superfamily. They are kind of transmembrane proteins which are located at chloroplast thylakoid membrane. They have been found in organisms such as cyanobacteria, bryophytes, higher plants, etc. It is initially found in the process of etiolated pea seedlings turning green. Due to being induced earlier than other light-induced proteins, they are named as ELIPs. They have the characteristics of transient accumulation and disappear once after the photosystem is completely developed. In this research, we analysized the ELIP gene family in Syntrichia caninervis based on the transcriptom data and screened out two ScELIP genes which have complete ORFs. We studied the bioinformatic characteritics, expression patterns and functions of these two ScELIP genes. The results were as follows: 1. Thirty nine annotated ScELIP unigenes were obtained in this research. Then we identified 27 Unigenes as ScELIP genes among which 6 genes have complete ORFs and chlorophyll-a/b binding domains. 2. Two ScELIP genes (ScELIP1 and ScELIP2) were screened out and their bioinformatic properties were analyzed. The results showed that ORFs of ScELIP1 and ScELIP2 contained 711bp and 624bp and they encoded 236 and 207 amino acids, respectively. Both of them contained a complete Chloroaophyll a/b-bind functional domain. Subcellular localization prediction revealed that the two ScELIPs were both located at the thylakoid membrane of chloroplast. Secondary structure prediction demonstrated that they both had three transmembrane helixes. Protein sequence alignment and phylogenetic analysis revealed that ScELIP1 had the closest relationship with moss and green alga, while ScELIP2 was closer to higher vascular plants, which showed the evolutionary trend clue from moss to vascular plants. 3. On the basis of bioinformatics analysis, we cloned these two ELIPs of S. caninervis and analyzed the expression patterns of ELIPs under various abiotic stresses and different light conditions. The results showed: ①ScELIP1 responded to all kinds of stresses such as slow dry, rapid dry, salt, cold, heat, while ScELIP2 didn’t respond to rapid dry. The reactions of both genes under NaCl, cold stress were fast and durable. ②They both had responses to UVA and UVB. However, ScELIP1 had a weak and transient response to UVA and ScELIP2 responded strongly and enduringly. Blue light could induce the expression of ScELIPs and enhance the induction of red light; On the contrary, red light inhibited the induction of blue light. The transcription levels of ScELIP1 and ScELIP2 increased with the increasing of light intensity. 4. We used genetically modified (GM) yeast to verify the functions of ScELIPs. By comparing the growth condition of GM yeast and control yeast under 20% PEG, 5mol/L NaCl, 53 ℃, -20 ℃ and UV stress, we finally found that GM yeast and the control showed the same growth condition. So the GM yeast system was not qualified to validate the functions of stress-resistance and photoprotection of ELIPs. Then we choosed model plant Arabidopsis to testify the function of ScELIPs. After exposed the transgenic and wild type Arabidopsis thaliana to high light, UV, red light, blue light, dry, salt, cold and heat stress, we observed the growth conditions and measured the related physiological indexes of them. Ultimately, we came to the conclusion that the two ScELIP2 gene was stress resistant and can help to avoid photooxidation.
学科领域植物学
语种中文
文献类型学位论文
条目标识符http://ir.xjlas.org/handle/365004/14684
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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周雅. 齿肋赤藓早期光诱导蛋白基因ELIP的克隆及功能分析[D]. 北京. 中国科学院大学,2015.
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