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齿肋赤藓四个 ScDREBs 基因特性及抗逆功能分析
梁玉青
学位类型硕士
导师管开云 ; 张道远
2017-05-01
学位授予单位中国科学院大学
学位授予地点新疆乌鲁木齐
学位专业理学硕士
关键词DREB转录因子 齿肋赤藓 胁迫抗性 功能验证 DREB transcription factor Syntrichia caninervis stress tolerance functional verification
摘要DREB transcription factor, belonging to the AP2 / EREBP family, is specificallypresent in plants and can regulate the expression of a series of downstream genes related todrought, salinity, low temperature and high temperature stresses, which plays vital roles inresponse to external adverse environmental factors, and are extensively investigated invarious plants. Syntrichia caninervis Mitt., an extremely dessication tolerance moss, isconsidered as a good candidate species for gene mining because of its outstanding drought,heat and UV resistant tolerance. In the past research, we build dehydration-rehydrationtranscriptome profile of S. caninervis and screened DREB family genes, and analyzed ongene expression pattern and gene abiotic function by yeast system of ten ScDREBs. In thisstudy, we selected four potential key ScDREBs genes and delve on further stress tolerancefunctional in Arabdopsis system. The work will shed light on the extremely stress tolerancemechanism of the moss, and will benefit for further development and utilization of thegene resources in crop breeding. The main conclusions are as follows:(1) Subcellular location assay of four ScDREBs proteins showed that all the fourScDREBs were located in both the nucleus and the cytoplasm of the onion or tobaccocells;(2) Transactivation activity assay of four ScDREBs proteins showed that ScDREB10,ScDREB3and ScDREB6 had transactivation activity except that ScDREB8 showed notransactivation activity;(3) Four ScDREBs genes were stably transformed into Arabidopsis by floral dipmethod, and the positive T3 generations were obtained for further gene function validation.Under the drought, salt, cold and heat treatment, the phenotypic change (root length andfresh weight) were observed, the physiological response (ROS level, determination ofantioxidant enzymes and proline content) were monitored and downstream gene expressionpattern were detected. The results showed that:Under the drought, salt and heat treatment, the Overexpressing-ScDREB10 lines had significantly higher seed germination rate and seedlings survival rate of transgenic linesthan wild type (WT). The transgenic lines also showed lower ROS level and higher SODand POD activities than those of WT at seedling stage. In addition, the expression ofScDREB10 gene could positively regulated a series of downstream stress toleranceassociated genes expression, which indicated that overexpressing-ScDREB10 increasedrought and salt tolerance in Arabidopsis; Under the salt treatment, the germination rate and seedlings survival rate ofoverexpressing-ScDREB8 gene lines were significantly higher than WT. The level of ROSin transgenic lines was significantly lower than that in WT, and the activity of antioxidantenzyme (SOD、POD) and proline content were significantly higher at seedling stage. Inaddition, ScDREB8 positively regulated a series of downstream stress tolerance associatedgenes expression. All results indicated that ScDREB8 increased salt tolerance inArabidopsis; Under the drought, high salt, cold and heat treatment, the overexpressing-ScDREB3lines had no significantly higher seed germination rate than the WT, but the survival ratewas significantly higher than that of the wild type under the extreme cold treatment(-20 ℃)at the seedling stage, showing that overexpressing-ScDREB3 gene might increase coldtolerance in Arabidopsis;Under the drought, high salt treatment, ScDREB6 transgenic lines showed nosignificant differences compared with the wild type, indicating that ScDREB6 might haveno abiotic stress tolerance ability. While the transgenic Arabidopsis showed significantlonger hypocotyl and a faster growth cycle phenotype at the seedling stage, implyingstrong hypocotyl elongation and growth-promoting function.
其他摘要DREB 转录因子,属于 AP2/EREBP 家族,特异的存在于植物中,能够调节下游一系列与干旱、盐碱、低温和高温等多种胁迫相关的基因的表达,在响应外界不利环境因素起着至关重要的作用,是目前研究的最广泛的一类植物抗逆基因。齿肋赤藓属于荒漠藓类,能够适应长期干旱、高盐、高温等极端环境,其体内抗逆基因资源丰富,是基因挖掘的好材料。项目组前期研究中,基于齿肋赤藓干燥-复水过程全局转录组数据库对整个 DREB 基因家族进行了分析与筛选,并对其中 10 个全长 ScDREB 基因进行了表达模式分析及酵母体系的功能验证。本研究拟进一步对上述筛选得到的四个重点的 ScDREBs 基因(ScDREB10、ScDREB8、ScDREB3、ScDREB6)进行基因特性和抗逆功能分析。本研究为开发利用该抗逆基因资源,定向培育抗逆植物新种质提供理论基础。主要结论如下:1.四个 ScDREBs 蛋白的亚细胞定位研究表明,四个 ScDREBs 蛋白均定位于细胞核和细胞质;2.四个 ScDREBs 转录因子的转录自激活研究表明:除 ScDREB8 基因没有转录自激活能力外,其余三个基因 ScDREB10、ScDREB3、ScDREB6 基因均具有转录自激活能力;3.利用浸花法将四个 ScDREBs 基因稳定转化模式植物拟南芥,得到阳性的 T3代转基因拟南芥种子,并在干旱、高盐、冷和热四种胁迫下,从表型观察(根长、鲜重等)、生理层面(ROS 水平、抗氧化酶和脯氨酸含量的测定)及分子水平上进行抗逆功能验证,所得到的结果如下:在干旱、盐和热处理下,转 ScDREB10 基因拟南芥萌发率显著高于野生型,在幼苗期转基因拟南芥的 ROS 水平相对于野生型更低,且 SOD、POD 活性较野生型高,同时激活了一系列下游抗逆基因的表达,在成苗期转 ScDREB10 基因拟南芥在干旱、盐和热处理下存活率显著高于野生型,综上,过表达 ScDREB10 基因能够提高转基因拟南芥的抗旱和抗盐能力;在盐处理下,转 ScDREB8 基因拟南芥萌发率显著高于野生型,幼苗期 ROS 水平显著低于野生型,而抗氧化酶(SOD、POD)活性及脯氨酸含量均显著高于野生型,并且 ScDREB8 激活了一系列相关抗逆基因的表达,成苗期转 ScDREB8 基因拟南芥在盐胁迫下的存活率显著高于野生型,综上,过表达 ScDREB8 基因能够提高转基因拟南芥的抗盐能力;在干旱、高盐、冷和热处理下,转 ScDREB3 基因植株萌发率与野生型比较没有显著性差异,但是在幼苗期的极端冷(-20 ℃)处理下的存活率显著高于野生型,综上,过表达 ScDREB3 基因可能具有提高转基因拟南芥的抗冷能力;在干旱、盐和热处理下,转 ScDREB6 基因植株抗性与野生型相比差异不明显,说明该基因可能不具有抗非生物胁迫能力,但转 ScDREB6 基因拟南芥表现出显著的茎杆增长表型,成苗阶段表现出更快的生长周期,初步研究表明该基因具有促进下胚轴伸长及促进植株生长发育的功能。
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/365004/14885
专题研究系统_荒漠环境研究室
作者单位中国科学院新疆生态与地理研究所
第一作者单位中国科学院新疆生态与地理研究所
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梁玉青. 齿肋赤藓四个 ScDREBs 基因特性及抗逆功能分析[D]. 新疆乌鲁木齐. 中国科学院大学,2017.
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