【作者】 艾兴辉；

【导师】 夏光敏；

【作者基本信息】 山东大学 ， 细胞生物学， 2013， 博士

【摘要】 盐、旱胁迫严重影响作物产量,因而分离耐逆相关基因、培育耐逆作物新品种非常重要。本实验室利用不对称体细胞杂交创制渐渗系技术,培育出了普通小麦(Triticum aestivum L.2n=42) JN177和长穗偃麦草(Thinopyrum ponticum2n=70)的小麦渐渗系耐盐、抗旱新品种山融3号(SR3)。SR3基因组发生了高频率的遗传和表观遗传学变异,是分离耐逆相关基因的优良材料。离子转运蛋白负责离子的吸收、外排或者转运,是植物响应盐、旱胁迫的重要组分。实验室前期通过组学分析,发现SR3的耐逆能力与其较强的离子平衡重建能力密切相关。本论文根据转录组分析结果,克隆了两个在SR3及其亲本JN177间差异表达的离子转运体基因,即负责丙酮酸转运的TaBASS2及负责钾离子转运的TaHAK11;初步分析了它们的生物学功能及在非生物胁迫下的作用机制。1胆汁酸：钠离子共转运蛋白基因TaBASS2的功能研究TaBASS2编码一个胆汁酸：钠离子共转运蛋白,在叶片中定位在叶绿体膜上,而在根中可能定位于白色体等其它质体中。TaBASS2主要在地上部分表达,而根中表达量较低。SR3三叶期幼苗中,盐胁迫下TaBASS2在叶片中下调表达、根中上调表达,旱处理下在根和叶中都下调表达,ABA处理后根和叶片中都是上调表达的,而过氧化氢处理下仅在根中上调表达。TaBASS2在酵母中不能转运Na+和丙酮酸,而在拟南芥中则能介导丙酮酸盐的跨叶绿体膜转运。TaBASS2的异源过表达提高了拟南芥AtNHD1的表达水平,推测TaBASS2转运丙酮酸依赖NHD1。TaBASS2过表达不影响拟南芥的种子萌发率,但抑制了子叶张开率以及幼苗的生长。研究表明,叶绿体逆行信号途径参与了幼苗早期发育调控。我们发现,TaBASS2过表达不影响MEcPP以及PAP信号途径关键组分AtHPL以及AtSAL1的表达；达草灭及洁霉素处理后,TaBASS2过表达系中四毗咯途径及PGE途径中下游应答基因AtLHCB1.1及AtLHCB2.4的表达被抑制程度明显降低。结果表明,TaBASS2可能通过抑制质体逆行信号转导中的四吡咯及PGE途径影响幼苗发育,而与MEcPP以及PAP信号途径无关。NaCl及ABA处理下,TaBASS2过表达系与Col-0的种子萌发率没有差异。TaBASS2过表达降低了子叶张开期幼苗对NaCl的耐受性、提高了对ABA的敏感性。但TaBASS2增强了拟南芥幼苗对盐胁迫、渗透胁迫和氧化胁迫等非生物胁迫的抗性以及对外源ABA的耐受性。我们发现,ABA合成途径中的关键酶AtAAO3、 AtNCED3以及ABA依赖的胁迫应答途径中AtMYC2、AtRD29B、AtRD22和AtRAB18表达也显著上调,表明TaBASS2可能通过促进ABA的合成及信号转导途径影响植株对非生物胁迫的响应。我们发现,TABASS2过表达株系中H202与’02含量明显提高,H202应答基因AtFER1及102应答基因AtAAA-ATPase和AtBAP1表达明显上调,ROS清除酶CAT活性及AtAPX2和AtCAT1表达也明显增强,表明H2O2及1O2参与了TaBASS2过表达株系对非生物胁迫的响应。总之,TaBASS2通过促进ABA生物合成与信号途径以及增强ROS信号途径提高拟南芥植株对非生物胁迫的抗性。至于子叶张开期幼苗应答非生物胁迫的机制,有待进一步研究。2高亲和性钾离子转运蛋白基因TaHAK11的功能研究TaHAK11是属于KT/KUP/HAK转运蛋白家族簇Ⅲ的成员,定位于细胞膜。我们共克隆到三个KaHAK11拷贝,包括JN177的TaHAK11-2及SR3的TaHAK11-3和TaHAK11-5,均定为于小麦2DL上。TaHAK11-5与TaHAK11-2存在一个L50H的变异,TaHAK11-3第50位氨基酸与TaHAK11-2相同；TaHAK11-3与TaHAK11-2/TaHAK1-5存在P175L、G352D、L398F、V585M四个氨基酸变异,其中前三个氨基酸在TaHAK11-3和其他植物HAK11亚簇成员中完全一样。结合TaHAK11的基因组定位特征,我们推测TaHAK11-3是植物HAK11的直系同源基因,而TaHAK11-2/TaHAK11-5是小麦多倍体进化过程中新产生的HAK11旁系同源基因,而TaHAK11-3在体细胞杂交及渐渗系形成过程中被激活表达。在酵母钾离子吸收缺陷突变体WΔ3中进行的功能互补实验表明,TaHAK11-2和TaHAK11-5介导高亲和性的K+吸收,而TaHAK11-3介导低亲和性的K+吸收；TaHAK11-3亲和性的改变与V585M的变异密切相关。TaHAK11-3在小麦地上部分表达量较高,而根中表达量较低；NaCl、PEG及外源ABA处理下,TaHAK11-3在叶中下调表达,而在根中上调表达。TaHAK11-3启动子-GUS转基因拟南芥的GUS染色显示,TaHAK11-3在叶片和茎中大量表达,而气孔保卫细胞中表达量也较高。钾饥饿条件下,与Col-0相比,TaHAK11-3过表达拟南芥叶片受损伤程度更严重。TaHAK11-3过表达降低了负责根中高亲和性K+吸收的AtHAK5的表达量,而提高了负责地上部K+外排的AtKUP2、AtKUP6、AtKUP8、AtGORK以及参与K+从地上部回流到根的AtAKT2的表达量。结果表明,TaHAK11-3通过限制根中K+吸收,增加地上部分K+外排以及增强K+通过韧皮部的回流降低对钾饥饿的耐受性。TaHAK11-3过表达提高拟南芥的抗旱性。与Co1-0相比,TaHAK11-3过表达系气孔开度减小,气孔保卫细胞K+外排相关基因AtKUP6、AtKUP8和AtGORK及ABA合成途径关键酶AtABA1、AtABA2的表达明显上调,而ABA信号通路抑制基因AtAB11表达则明显下调。结果显示,TaHAK11-3通过促进ABA合成和信号途径以及保卫细胞钾离子外排,降低气孔开度来提高拟南芥的抗旱性。更多还原

【Abstract】 Salinity and drought stress adversely influenced the productivity of crops, so it is urgent to isolate stress tolerance related genes for cultivating new crop varieties with high stress tolerance. In our lab, wheat introgression cultivar ShanRong No.3(SR3), with high productivity and strong salt and drought tolerance, was bred from hybrids of common wheat JN177(Triticum aestivum L.2n=42) and tall grass(Thinopyrum ponticum2n=70) using asymmetric somatic hybridization method. High frequency of genetic and epigenetic variation has taken place in SR3genome, which make the cultivar an improved variety for isolating stress tolerance related genes.Ion transporters function in ionic uptake, excretion or transport, and are pivotal components in response to salt and drought stress in plants. In the previous work of our lab, stress tolerant capability in SR3had been found to be closely related to its superior ability in ion homeostasis reconstruction. In this study, we cloned two transporter genes, TaBASS2involved in pyruvate transport and TaHAK11responsible for K+transport, according to their differential expression patterns between SR3and JN177according to the transcriptomical data, and analyzed their biological functions and mechanisms in response to abiotic stresses.1. Functional analysis of a bile acid:Na+symporter gene named TaBASS2TaBASS1encoded a bile acid:sodium symporter, which was located on chloroplast envelope in leaf while in other plastids, such as leucoplast, in root.. TaBASS2transcribed highly in shoot while lowly in root. In SR3seedlings at three-leaf stage, TaBASS2expression was down-regulated in leaf and up-regulated in root under salt stress, and down-regulated both in root and leaf under drought stress. TaBASS2transcripts increased both in root and leaf when applied with exogenous ABA, and increased only in root after H2O2treatment.TaBASS2could not transport Na+and pyruvate in yeast. However, it enhanced the transport of pyruvate across the chloroplast envelope in Arabidopsis. Ectopic expression of TaBASS2in Arabidopsis raised the expression level of AtNHD1, suggesting that transport of pyruvate by TaBASS2depends on NHD1.TaBASS2overexpression didn’t influence seed germination rate, but inhibited opening rate of cotyledons and the growth of seedlings at germination period in Arabidopsis. Previous studies have showed that retrograde signaling pathways are involved in early seedling development. We found TaBASS2overexpression didn’t influence the expression of AtHPL and AtSAL1, key components in MEcPP and PAP signaling pathway, respectively. TaBASS2overexpression attenuated the inhibition effect of norflurazone and licomycin on the expression of AtLHCB1.1and AtLHCB2.4, downstream responsive genes in tetrapyrrole and PGE plastid retrograde signaling pathway. Above data demonstrate that TaBASS2may restrict seedlings development by inhibiting tetrapyrrole and PGE plastid retrograde signaling pathway rather than MEcPP and PAP signaling pathway.There was no difference in seed germination rate between TaBASS2overexpression lines and Col-0after treatment with NaCl and ABA. TaBASS2overexpression decreased tolerance to NaCl and increased sensitivity to ABA of seedlings at cotyledon opening stage, but its overexpression enhanced tolerance to salinity, osmotic and oxidative stresses and lowered sensitivity to exogenous ABA in Arabidopsis seedlings at post-germination stage. We found the contents of H2O2and1O2increased, the expression of H2O2responsive gene AtFER1as well as1O2responsive gene AtAAA-ATPase and AtBAP1obviously up-regulated, and the activity of ROS scarvenging enzyme CAT and the expression of AtAPX2and AtCAT1significantly raised. In summary, TaBASS2improved tolerance to abiotic stress by promoting ABA biosynthetic and signaling pathway and altering ROS homeostasis. The mechanism underlining the response to abiotic stress at cotyledon opening stage deserves further study.2. Functional analysis of a high affinity K+transporter gene TaHAK11TaHAK11belonged to cluster Ⅲ of KT/KUP/HAK transporter family and was located on cytoplasmic membrane. Here, we cloned three TaHAK11copies totally, including TaHAK11-2from JN177, TaHAK11-3and TaHAK11-5from SR3, which were all located on wheat chromosome2DL. There has one point mutant of L50H between TaHAK11-5and TaHAK11-2, four mutants, P175L, G352D, L398F, V585M between TaHAK11-3and TaHAK11-2/TaHAK11-5. As for the four allelic variation sites, the first three amino acids are the same among TaHAK11-3and TaHAK11subcluster members in other plants. In line with the genomic locating features of TaHAK11, we postulated that TaHAK11-3is a homologous gene of plant HAK11while TaHAK11-5/TaHAK11-2is a new HAK11paralogous gene produced during polyploid evolution of bread wheat, and TaHAK11-3maybe be activated during somatic hybridization and development of introgression lines. Functional complementation experiments in K+uptake deficient yeast mutant showed that TaHAK11-2and TaHAK11-5mediate high affinity K+uptake while TaHAK11-3mediates low affinity K+uptake. The difference in affinity to K+uptake may be due closely to the variation of V585M.The transcript aboundance of TaHAK11-3was high in shoot and low in root of wheat at three-leaf stage. When treated with NaCl, PEG and exogenous ABA, the transcription level of TaHAK11-3decreased in leaf while increased in root. GUS staining of TaHAK11-3promoter-GUS construct transgenic Arabidopsis denoted that TaHAK11-3was expressed highly in leaf, stalk and guard cells.TaHAK11-3overexpression in Arabidopsis pushed leaf damage during K+deprivation. Its overexpression decreased the expression of A/HAK5which is responsible for high KT uptake in root and increased that of AtKUP2, AtKUP6, AtKUP8, AtGORK which are involved in K+exclusion in shoot and AtAKT2which is responsible for recirculating of K+from shoot to root. Studies above demonstrated that TaHAK11-3lowerred the tolerance to K+deprivation via the restriction for K+uptake in root, increasement of K+exclusion in shoot and promotion of K+recirculation from shoot to root.TaHAK11-3overexpression enhanced tolerance to drought. The overexpression promoted stomatal closing, improved the expression of AtKUP6, AtKUP8and AtGORK associated with K+exclusion from guard cells and AtABA1, AtABA2 encoding key enzymes of ABA biosynthetic pathway, restricted the transcription of AtAB11, the negative regulatory component of ABA signaling pathway. These results showed that TaHAK11-3improved tolerance to drought through promoting stomatal closure by promoting ABA biosynthesis and signaling pathway and K+exclusion from guard cells.更多还原