【作者】 向建华；

【导师】 陈信波；

【作者基本信息】 湖南农业大学 ， 生物化学与分子生物学， 2013， 博士

【摘要】 高温、低温、干旱和盐碱等非生物胁迫严重影响植物的生长发育,导致农作物的产量和品质降低。对植物逆境相关基因功能的研究,可为农作物的耐逆定向育种提供理论基础。植物存在可遗传的钾营养效率差异,提高农作物自身钾营养效率是目前农作物品质遗传改良的重要研究方向。本研究利用突变体、过量表达和遗传互补等技术手段对拟南芥UNC-93基因进行了功能鉴定,初步鉴定出在提高植物耐逆性、促进钾在植物体根冠部的运输和促进植株生长等方面发挥重要作用。主要结果如下：1.根据水稻高温胁迫下表达谱分析数据,共筛选了11个高温显著诱导表达基因,并从ABRC获得了其拟南芥同源基因的突变体。经PCR和RT-CR检测,共得到11个纯合突变体。2.通过对拟南芥11个突变体在高温、高盐、渗透和干旱等胁迫处理下耐受能力检测,筛选出2个对胁迫耐受能力显著降低的突变体unc-93-1和unc-93-2。unc-93-2株型矮小、开花发育较迟,而突变体unc-93-1在株型上与野生型的差异不及突变体unc-93-2明显。RT-PCR检测表明unc-93-1有UNC-93基因的3’端(T-DNA插入点之后)非全长转录产物表达,而unc-93-2却没有。3.UNC-93是钾离子通道的调控蛋白。UNC-93蛋白的跨膜结构分析表明其具有10个跨膜结构,预测为膜蛋白。亚细胞定位研究证实了UNC-93的确为膜蛋白。通过Real-time PCR检测,UNC-93基因的表达可被高温、高盐、甘露醇和ABA等处理诱导上调,但是对低温没有明显响应。正常生长条件下在各组织中均能检测到UNC-93的表达,但是根和幼苗中表达最强烈。经GUS报告基因转基因拟南芥材料的GUS组织化学染色发现,GUS在根、幼苗和叶脉中活性最强。4.构建了利用CaMV35S强启动子驱动UNC-93全长cDNA的过量表达载体和UNC-93基因3’端部分cDNA的过量表达载体,经农杆菌介导的花序浸泡法转化到野生型拟南芥(Col-0)中得到过量表达株系,转化到突变体UNC-93-1和UNC-93-2中得到遗传互补株系。5.突变体unc-93-1和unc-93-2在幼苗生长阶段都表现出明显降低对高温、高盐、干旱、甘露醇和ABA等处理的耐受能力,而UNC-93全长cDNA的过量表达能显著提高植株的耐盐性和耐旱性。在低温条件下,突变体lnc-93-1. unc-93-2.UNC-93过量表达和野生型没有明显差异。主要逆境相关基因RD29A. RD22、DREB2A.COR15A、COR47、HsfA2、Hsp101和Hsp70在胁迫处理前或者胁迫处理后突变体中表达量明显低于野生型,而UNC-93过量表达植株中部分基因的表达量却高于野生型。ABA响应基因AB11、AB12、MYB2和SnKR2.6等在突变体、UNC-93过量表达和野生型中的表达量具有显著差异。推测UNC-93可能是通过依赖于ABA途径来调控植物的耐逆性。在种子萌发阶段,unc-93-2和UNC-93过量表达种子的萌发相比野生型均受到高盐、渗透、高温和ABA的强烈抑制,而unc-93-1相比野生型却降低了对胁迫的敏感性。6.对突变体、UNC-93过量表达和野生型钾含量测定显示,突变体unc-93-1和unc-93-2冠部的钾含量都低于野生型。UNC-93过量表达植株只在低钾条件下钾含量才显著高于野生型,而在正常钾条件下,与野生型没有明显差异。根部钾含量各株系均无明显差异。正常生长条件下,unc-93-2的生长发育受到抑制,unc-93-1也表现出生长延迟现象,UNC-93过量表达植株相对野生型株型高大,根系发达。低钾条件下,与野生型和UNC-93过量表达相比,突变体unc-93-1和unc-93-2在幼苗生长上受到更严重抑制,而且黄化。在低钾低铵条件下,突变体unc-93-1和unc-93-2萌发后表现出子叶褐化、根弯曲多根毛等现象。7.遗传互补实验表明,当把UNc-93全长cDNA的过量表达载体转化到突变体unc-93-1和unc-93-2中,突变体unc-93-1和unc-93-2所具有的对高盐、渗透等胁迫的敏感性和unc-93-2的变异株型特征都得到明显的修复。而当UNC-93基因3’端部分cDNA的过量表达载体转化到unc-93-2以后,也能在一定程度上修复unc-93-2矮小的特征。更多还原

【Abstract】 The plant growth and development were greatly affected by abiotic stresses, such as high or low temperature, drought and salinity, and resulted in significant reductions in crop yields and quality. Research on the functions of stress related genes in plant can provide theoretical basis for targeted genetic engineering of high stress tolerant crops. Plant K+efficiency can be genetically controlled, and diverse plant species have different K+efficiency. High K+efficiency is a target in genetic improvement of crop quality. The objective of this research is to characterize the functions of Arabidopsis UNC-93in stress tolerance, potassium transport and plant growth. The main results are as follows:1. A total of11highly heat responsive genes were screened from rice microarray data and19mutants of their homologous genes in Arabidopsis were obtained from ABRC. A total of11homozygous lines were identified by PCR and RT-PCR.2. The mutant unc-93-1and unc-93-2showed significantly decreased tolerance to high temperature, salt and osmotic stress among the11Arabidopsis mutants screened. The unc-93-2showed dwarf and later anthesis, while unc-93-1had less significant difference in plant type compared with wild type (WT) than that of unc-93-2. A truncate transcript of3’ end of UNC-93(behind the site of T-DNA) was detected in unc-93-1by RT-PCR analysis, but no any expression of UNC-93was detected in unc-93-2.3. UN-93is a K+channel regulatory protein. UNC-93is a membrane protein with10transmembrane helices. Transient expression assay located UNC-93at the plasma membrane in onion epidermal cells. The expression of UNC-93was induced by high temperature, salt, osmotic and abscisic acid (ABA) treatments. But there was no response to low temperature. Although the transcript of UNC-93could be detected in almost all tissues, it was higher in root and seedling. The activities of GUS were higher in root, seedling and vein by GUS assay in UNC-93promoter driven GUS report gene transgenic plants.4. The overexpression vectors of full or partial cDNA of UNC-93were constructed, and were separately transformed into WT (Col-0), unc-93-1or unc-93-2.5. The mutant unc-93-1and unc-93-2greatly decreased stress tolerance to high temperature, salt, drought, osmotic and ABA compared with WT. Overexpression of UNC-93significantly improved to salt and drought tolerance. No significant difference was observed between unc-93-1, unc-93-2UNC-93-overexpressing plants and WT under low temperature treatment. The expression levels of main stress related genes were significantly decreased in mutants but were upgraded in WVC-93-overexpressing plant. The expression levels of ABA-responsive genes, such as ABI1, ABI2, MYB2, SnKR2.6, had significantly differences among mutants, WVC-93-overexpressing plants and WT. Seed germination of unc-93-2and WVC-93-overexpressing line were greatly inhibited compared with WT under high temperature, salt, osmotic and ABA treatments, while unc-93-1mutant decreased sensitivity to these stresses compared with WT.6. Compared with WT, the K+contents in shoots of unc-93-1and unc-93-2were significantly decreased, WVC-93-overexpressing line had much higher K+contents under low K+concentration, but there were no obvious differences between WVC-93-overexpressing line and WT under normal K+concentration. There were no detectable changes of the K+contents in roots of unc-93-1, unc-93-2, WVC-93-overexpressing line and WT. Under normal condition, compared with WT, the growth and development of unc-93-2was inhibited, and the growth of unc-93-1was delayed, while WVC-93-overexpressing plants showed higher shoots and more roots. Under low potassium condition, compared with WT and WVC-93-overexpressing plants, the growth of unc-93-1and unc-93-2were inhibited more gravely and etiolation. The unc-93-1and unc-93-2showed brown cotyledons, curled principal roots and more lateral roots after seeds germination under low K+and NH4+concentration.7. The genetic complement assay showed that overexpression full cDNA of UNC-93can resume the tolerance to salt and osmotic stresses in unc-93-1and unc-93-2. The dwarf phenotype of unc-93-2returned to normal. When the overexpression vector of partial cDNA of UNC-93was transformed into unc-93-2, the dwarf phenotype of unc-93-2also returned to a certain extent.更多还原