沙冬青种质资源与抗旱基因研究

【作者】 刘冉；

【导师】 周宜君；

【作者基本信息】 中央民族大学 ， 植物学， 2012， 硕士

【摘要】 蒙古沙冬青(Ammopiptanthus mongolicus)主要分布在我国西北地区的沙漠、荒漠区,是该区唯一的常绿阔叶灌木,具有很强的抗旱、抗寒等抗逆性。本研究以蒙古沙冬青(A. mongolicus)幼苗为实验材料,测定干旱胁迫下沙冬青幼苗叶片和根生理生化指标；采用454高通量测序技术建立了沙冬青根转录本数据库,并进行了相关分析；采用qRT-PCR技术检测沙冬青抗旱相关基因的表达模式,以揭示沙冬青抗旱生理基础和分子机制。结果如下：采用20%PEG6000胁迫处理1h-72h,沙冬青幼苗叶片的RWC、MDA含量和REL与对照相比没有变化；经过24h胁迫处理,沙冬青幼苗根的RWC与对照相比显著降低,而MDA含量和REL显著增加。胁迫处理6h,沙冬青幼苗叶片和根的脯氨酸含量均有大量积累。胁迫条件下沙冬青幼苗叶片的SOD活性表现为先降低后上升,POD活性略有增加,处理6h后CAT活性降低；沙冬青幼苗根中的SOD、CAT和POD活性随胁迫时间加长而增加,72h处理SOD和POD活性水平最高。上述研究结果说明,采用灌根法进行模拟干旱胁迫处理,叶片和根中的相关生理响应特征不尽相同,根的反应较强。应用454高通量测序技术得到沙冬青根转录组共672002条高质量(HQ)序列,平均长度279bp。拼接序列27366条,包括14912个序列重叠群(contig)和12454条singleton,总长约14Mb。共9771个蛋白序列获得了注释,采用GO功能富集分析分别从生物学过程、细胞组分和分子功能进行分类。并根据GO功能富集分析,选择了响应渗透胁迫、氧化胁迫、激素刺激和光信号刺激等4类共计27个抗旱相关基因,采用qRT-PCR技术,以20%PEG6000胁迫1h和72h的沙冬青根为材料分析了表达模式,各自响应的方式不同。以1h和72h胁迫处理皆为上调表达且相对表达量为2以上为标准确定为干旱胁迫诱导的基因,共计获得了4个基因,分别编码糖苷水解酶家族蛋白、过氧化物酶超家族蛋白、谷胱廿肽过氧化物酶(GPX3)和向光反应性NPH3家族蛋白；以1h和72h胁迫处理皆为下调表达且相对农达量为0.8以下为标准确定为干旱胁迫抑制的基因,共计获得了4个基因,分别编码脱水素家族蛋白(ERD14)、硫酸盐转运子(SULTR1:3)、硫氧还蛋白和向光反应性NPH3家族蛋白。本研究为深入开展沙冬青抗旱机理和抗旱基因的研究奠定了基础。更多还原

【Abstract】 As the only evergreen broad-leaf shrub in the northwestern desert of China, Ammopiptanthus mongolicus shows very strong resistance to drought and cold. In order to explore the physical and molecular mechanisms underlying the drought tolerance in A. mongolicus, we analyzed physiological and biochemical characteristics in A. mongolicus seedling under drought stress. We performed large-scale transcriptome sequencing of A. mongolicus root using Roche/454next-generation sequencing technology; and the drought responsive genes of A. mongolicus differentially expressed under drought treatment were identified by further quantitative real-time PCR analysis. The results are discussed as follow:The RWC, MDA and REL in the leaf of A. mongolicus seedling did not change compared with control under treatment of20%PEG6000for1h-72h. After24h drought stress, RWC of the root of A. mongolicus seedling decreased significantly, while MDA and REL increased. The proline accumulated abundantly in both leaves and roots of A. mongolicus after6h drought stress. In the drought condition, the activities of SOD, CAT and POD in A. mongolicus seedling were distinctly regulated. In leaves of A. mongolicus seedling, the activity of SOD was down regulated at first then increased; the activity of CAT decreased gradually, but the POD increased a little at6h drought treatment. In the root, the activities SOD, CAT and POD were all up regulated, among which SOD and POD increased the most. These results indicated that the physical responses were different in leaves and roots of A. mongolicus and the response of root was more intense, after watering root with20%PEG6000.The transcriptome of A. mongolicus root obtained672,002HQ reads from a454GS XLR70Titanium pyrosequencer, and the average length was279bp. The672,002preproeessed sequencing reads were assembled into27,366unique sequences including14,912contig and12,454singlets, which total length was approximately14Mb. Gene ontology assignments were used to classify the functions of the A. mongolicus transcripts, and the9,771annotated sequences can be categorized into3functional groups:biological process, cellular component, and molecular function. To identify drought responsive genes,27unigenes were selected from the unique sequences classified in GO categories by qRT-PCR technology. When the expression models were analyzed with the root of the A. mongolicus treated by20%PEG6000,27genes were identified and categorized into4groups by the stress they responsed to, namely osmotic stress, oxidative stress, hormone stimulus and light stimulus. A gene was difined as the drought stress induced gene if it was up-regulated under1h and72h stress and the relative expression exceeded2-fold change.4 genes we obtained met such a definition, coding glycoside hydrolase family2protein, peroxidase superfamily protein, GPX3and phototropic-responsive NPH3family protein; A gene was difined as the drought stress inhibited gene if it was down-regulated under1h and72h stress and the relative expression did not exceed0.8-fold change.4genes we obtained met such a definition, coding ERD14, SULTR1;3, thioredoxin family protein and phototropic-responsive NPH3family protein. This study facilitated the research on drought resistance mechanism and related genes in A. mongolicus.更多还原