西瓜交替氧化酶ClAOX及其相关基因ClCASPL在低温胁迫下的功能研究

发布时间：2018-03-22 09:00

本文选题：西瓜　切入点：低温胁迫　出处：《浙江大学》2016年博士论文　论文类型：学位论文

【摘要】：西瓜(Citrullus lanatus)是深受人们喜爱的水果之一,我国是世界上西瓜栽培面积和产量最大的国家。西瓜起源于非洲,喜温喜光,对冷害较为敏感。设施早春栽培的西瓜时常会遇到低温弱光的影响,造成幼苗长势弱和后期生长不良,严重影响西瓜的产量和品质。研究西瓜低温相关基因的功能可以为其耐低温分子育种提供理论基础。本文主要研究了西瓜交替氧化酶基因(ClAOX)及其多态性位点、凯氏带形成相关基因(CICASPL)在低温胁迫下的功能。本文采用简并引物和基因组数据库检索的方式证明西瓜基因组中仅存在一个编码交替氧化酶的基因。进化树分析发现该基因属于AOX2亚家族,基因结构分析发现该基因具有4个外显子和3个内含子。亚细胞定位、低温诱导表达及组织特异性表达研究表明,ClAOX定位于线粒体,能够被低温胁迫诱导,且在西瓜根、茎、叶、花、果实中具有不同的表达。CIA OX-promoter-gus转基因系GUS染色结果表明,不同组织器官不同发育阶段ClAOX表达不同。低温诱导表达及组织发育特异性表达结果证明,西瓜ClAOX基因同时具备了AOX1和AOX2两个亚家族的功能特征。对不同西瓜亚种、种间交替氧化酶基因克隆比对发现,不同基因型西瓜间交替氧化酶基因存在丰富的多态性。饲用西瓜和普通栽培西瓜之间仅存在一个单核苷酸多态性位点(K96、N96)。采用转基因的方法对该位点功能进行验证发现：10℃低温胁迫下,与ClAOXN相比ClAOXK具有更长的主根和更大的地上部鲜重,叶绿素荧光数据也表明CIAOXK比ClAOXN更好的耐低温特性。DAB、NBT染色显示低温胁迫后ROS积累ClA XN略多于ClAOXK。过氧化氢脱色试验发现低温预处理能够提高转基因植株对外源过氧化氢的抗性,10℃C条件下处理后的植株抗性最强,15℃次之,正常条件下生长的植株抗性最弱。各低温胁迫下ClA XK对外源过氧化氢的抗性均优于ClAOXN。这些结果表明,ClAOXK和ClAOXN对低温抗性的差异可能与ROS信号通路密切相关。西瓜ClCASPL是从SHAM预处理后低温胁迫下的西瓜叶片抑制性差减杂交文库中筛选出的未知功能蛋白,该基因的同源基因AtCASPL4C1属于CASP (UPF0497)基因家族。对西瓜中ClCASP家族研究发现,该家族具有30个成员,分布于不同的染色体上。组织表达检测发现不同家族成员在根、茎、叶花、果实具有不同的表达模式。亚细胞定位研究发现,ClCASPL定位于细胞膜上；GUS染色结果表明,ClCASPL同源基因AtCASPL4C1普遍表达于各组织器官中,且能被低温诱导表达。凯氏带染色发现AtCASPL4C1缺失或者ClCASPL基因过表达均未影响根系凯氏带的形成。低温诱导表达发现,ClCASPL及其同源基因AtCASPL4C1均能够被低温胁迫不同程度诱导表达。采用拟南芥转基因系研究发现,ClCASPL及其同源基因AtCASPL4C1负调控植株的发育和低温抗性。采用拟南芥突变体研究发现AtAOX2基因缺失能够显著抑制ClCASPL同源基因AtCASPL4C1在叶片中的表达。通过对西瓜AOX和CASP两个家族基因功能的研究不仅能够为西瓜耐低温育种提供理论基础,同时也能将交替氧化途径与凯氏带发育及功能发挥相关联。
[Abstract]:Watermelon (Citrullus lanatus) is one of the favorite fruit is watermelon, China's cultivated area and output of the largest country in the world. Watermelon originated in Africa, warm like light is sensitive to chilling injury, early spring cultivation of watermelon. The facility often encountered in low temperature and weak light, resulting in weak seedling growth and late growth, seriously affect the yield and quality of watermelon watermelon. Low temperature related genes can provide a theoretical basis for the molecular breeding of low temperature resistance. This paper mainly studies the watermelon alternative oxidase gene (ClAOX) and its related gene polymorphism sites, formation of Casparian strips (CICASPL) under low temperature stress. This paper using degenerate primers and genome database search. That there is only one alternative oxidase gene encoding the watermelon genome. Phylogenetic analysis showed that this gene belongs to AOX2 subfamily, gene structure Analysis showed that this gene has 4 exons and 3 introns. The subcellular localization of low temperature induced expression and tissue specific expression studies showed that ClAOX was localized to the mitochondria, can be induced by low temperature stress, and the watermelon root, stem, leaf, flower,.CIA OX-promoter-gus transgenic GUS staining indicated that the expression is different in the fruit, different expression in different organs at different developmental stages of ClAOX. Low temperature induced expression and tissue specific expression of development results show that watermelon ClAOX gene simultaneously has the function characteristics of AOX1 and AOX2 two subfamilies. The different subspecies of watermelon, interspecific alternative oxidase gene cloning than the discovery of alternative oxidase gene polymorphism of the rich the different genotypes of watermelon. Only a single nucleotide polymorphism between forage and cultivated watermelon watermelon (K96, N96). The work method of transgenic loci To verify the findings: 10 DEG C under low temperature stress, compared with ClAOXN, ClAOXK has a longer taproot and greater shoot fresh weight, chlorophyll fluorescence data also show that CIAOXK is better than ClAOXN in the low temperature resistant properties of.DAB, NBT staining showed that the ROS ClA XN after low temperature stress accumulated slightly more than ClAOXK. hydrogen peroxide bleaching test found that low temperature pretreatment can to improve the resistance of transgenic plants to exogenous hydrogen peroxide plant, the strongest resistance after treatment 10 DEG C under the condition of 15 DEG C, the second, the resistance was the weakest growth under normal conditions. The resistance of hydrogen peroxide under the low temperature stress ClA XK external source are better than those of ClAOXN.. These results indicate that the ClAOXK and ClAOXN difference of low temperature resistance may with the ROS signaling pathway. ClCASPL is closely related to watermelon watermelon leaves from SHAM pretreated under low temperature stress suppression subtractive hybridization screening of proteins of unknown function in the library. Homologous gene AtCASPL4C1 belongs to CASP (UPF0497) gene family. The research findings in watermelon ClCASP family, the family has 30 members, located in different chromosomes. The expression was found in different family members in the roots, stems, leaves, flowers, fruits with different expression patterns. The study found that the subcellular localization, ClCASPL localization in the cell membrane; GUS staining indicated that ClCASPL homologous gene AtCASPL4C1 is ubiquitously expressed in various tissues and organs, and can be induced by low temperature. The expression of Casparian strips were found missing AtCASPL4C1 or ClCASPL gene expression did not affect the root formation of Casparian strips. Low temperature induced expression of ClCASPL homologous gene and found that AtCASPL4C1 can expression was induced in different degrees. The low temperature stress in Arabidopsis transgenic lines showed that homologous ClCASPL gene AtCASPL4C1 and its negative regulation of plant development and low temperature resistance The study found. Arabidopsis mutant expression deletion of AtAOX2 gene can inhibit the ClCASPL homologous gene AtCASPL4C1 in the leaves. Based on the research of AOX and CASP two family watermelon gene function can not only provide the theoretical basis for the breeding of cold resistant watermelon, but also will alternate oxidation pathway and Kjeldahl and function associated with development.

【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S651

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