【作者】 张洪映；

【导师】 王成社；

【作者基本信息】 西北农林科技大学 ， 作物遗传育种， 2011， 博士

【摘要】 小麦是世界上主要的粮食作物之一,干旱、盐和极端气候等逆境因子是限制小麦产量和品质提高的重要因素。挖掘和利用小麦逆境应答基因资源是应用现代基因工程技术改良小麦抗逆性的前提和基础,对于深入了解植物抗逆机制具有重要意义。蛋白质的可逆磷酸化是在逆境条件下植物体内能量代谢和逆境信号传递的重要机制。据报道,蔗糖非发酵相关蛋白激酶家族2 (Sucrose non-fermenting1-related protein kinase 2, SnRK2)在逆境信号传递途径中具有重要作用。本研究通过筛选本实验室构建的小麦水分胁迫应答cDNA文库,获得小麦SnRK2.8基因的EST序列,通过电子克隆的方法获得了小麦TaSnRK2.8基因的全长cDNA序列。通过研究其表达特性、亚细胞定位、染色体定位、单核苷酸多态性和启动子序列,并结合转基因功能验证结果,初步揭示TaSnRK2.8在逆境胁迫下的表达模式、抗逆功能及可能的信号通道。主要研究结果包括:1.小麦TaSnRK2.8基因的cDNA序列全长为1431 bp,包含一个1101 bp的开放阅读框,编码含367个氨基酸的多肽。预测TaSnRK2.8蛋白同时存在丝氨酸/苏氨酸激酶和酪氨酸激酶催化结构域。氨基酸序列比对结果表明,TaSnRK2.8蛋白分子量约42 kDa,等电点为4.87,与玉米、水稻和拟南芥中的直系同源基因的相似性分别为94%、94.8%和76.5%。聚类分析结果表明,TaSnRK2.8基因属于SnRK2家族第三亚族成员。2.组织表达分析结果表明,TaSnRK2.8基因在小麦根部的表达量最高,其次为茎部,在叶片和穗中的表达量较低。TaSnRK2.8在胁迫处理下的表达分析结果表明,该基因受ABA、高盐、高渗和低温胁迫诱导表达,且在不同胁迫条件下基因的表达模式不同,其对各诱导条件的敏感度为:高渗>低温>ABA>高盐。3.亚细胞定位及转拟南芥检测结果均表明,TaSnRK2.8基因在细胞膜、细胞质和细胞核中均有分布。4.转基因功能验证结果表明,TaSnRK2.8过表达后可以促进植株根部的生长发育,增加细胞内的渗透物质,这些变化有利于根对土壤水分和养分的吸收,从而提高植物细胞膜在逆境胁迫条件下的稳定性,增强叶片的保水能力及光合能力等,进而提高植物的耐盐、抗旱和耐低温能力。此外,TaSnRK2.8过表达植物体内的可溶性糖含量显著降低,表明该基因在植物糖代谢过程中起重要作用。通过qRT-PCR研究TaSnRK2.8转基因株系中其它抗逆相关基因的表达量变化,结果表明,TaSnRK2.8转基因株系中的ABA合成相关基因(ABA1, ABA2)、ABA信号传递相关基因(ABI3, ABI4, ABI5)和逆境诱导基因(CBF1, CBF2, CBF3, RD20A, RD29B)均上调表达,其中CBF1、CBF2和CBF3为非ABA依赖型基因,RD20A和RD29B为ABA依赖型基因。5. TaSnRK2.8基因组序列长度长约6.1 kb,包含9个外显子、8个内含子。其在普通小麦基因组中至少存在两种基因型TaSnRK2.8-A和TaSnRK2.8-B。利用中国春小麦缺四体系将TaSnRK2.8-A定位于小麦5A染色体上。分析165份小麦材料TaSnRK2.8-A-C序列的单核苷酸多态性,共检测到3个核苷酸变异,全部为转换,无插入缺失(InDel),SNP的频率为1 SNP/250 bp。核苷酸多样性(π)为0.00068,基于分离位点数目的多样性θw值为0.00070。其中,3′端UTR区的核苷酸多样性最高,次之为内含子区,外显子区的核苷酸多态性最低。中性测验结果表明TaSnRK2.8-A-C序列符合中性进化,这些遗传变异可能是突变-遗传漂变的结果。TaSnRK2.8-A-C序列等位变异(5917 bp, A→G)与群体性状的关联分析结果表明,该基因与糖代谢相关,进而影响小麦株高、叶宽、生物量和抽穗期等生长发育相关的性状,同时也与抗旱指数相关。同时,就A/G等位变异位点而言,A基因型是一种优异的等位基因类型。6.分离了TaSnRK2.8基因上游1797 bp的启动子序列,该序列富含A/T碱基,如TATA-box、CAAT-box、ABRE、HSE、TC-rich repeats、LTR及C-repeat/DRE等顺式作用元件。通过对TaSnRK2.8启动子序列5′端四个连续缺失片段对GUS基因的驱动能力分析,发现只有Dp1870能够激活下游GUS的表达,且仅在转基因植株的叶柄和茎部表达,在叶片及根部均无表达。GUS荧光定量分析结果显示,在非胁迫条件下Dp1870驱动能力约为35S强启动子0.16~0.35倍。本文的研究结果表明,小麦蔗糖非发酵相关蛋白激酶TaSnRK2.8参与了植物体内糖代谢及抗逆信号传递途径,其过表达后可以显著增强植物的抗逆性。更多还原

【Abstract】 Drought, salinity and extreme temperatures are major factors limiting the yield and quality of wheat (Triticum aestivum L.). To survive adverse stresses, plants have developed complex signaling networks to perceive external stimuli, and then manifest adaptive responses at molecular and physiological levels. Reversible protein phosphorylation is central to the perception of, and response to, stress conditions. Sucrose non-fermenting1-related protein kinase 2 (SnRK2) plays a key role in abiotic stress signaling via phosphorylation in plants.To discover and utilize the genetic resources involved in abiotic stress tolerance, we constructed the cDNA library responsed to water defict from wheat seedlings using suppressive subtractive hybridization. In the study, one EST of SnRK2.8 was screened from the cDNA library. Based on the EST sequence, we isolated TaSnRK2.8 from wheat and characterized the functions in abiotic stress responses. The results were as follows:1. The full length cDNA of TaSnRK2.8 was cloned from wheat by in silico cloning. The TaSnRK2.8 cDNA was 1431 bp in length, which contained an ORF of 1101 bp and encoded a protein of 367 amino acids. The putative protein had a calculated molecular mass of 42 kDa and isoelectric point of 4.87. Moreover, TaSnRK2.8 might have both activities of serine/threonine and tyrosine kinases. Amino acid sequences alignment analysis showed that TaSnRK2.8 showed homology with counterpart SnRK2 family members from other plant species, viz. Oriza sativa, Zea mays and Arabidopsis thaliana. TaSnRK2.8 has 94.8% identity to OsSAPK8, 94% to ZmSAPK8, and 76.5% to AtSnRK2.2, respectively. Phylogenetic analysis indicated that TaSnRK2.8 belonged to subclass III subfamily of SnRK2.2. Quantitative real-time PCR were used to analyze the expression patterns of TaSnRK2.8 in wheat. TaSnRK2.8 was constitutively expressed in wheat, strongly in roots, weakly in stems, and marginally in leaves and spikes. Abiotic stress responses analyses revealed that TaSnRK2.8 was involved in response to PEG, NaCl and cold stresses, and possibly participates in ABA-dependent signal transduction pathways. The sensitivity degrees of TaSnRK2.8 responsing to abiotic stresses was in the order of hyperosmolality > low temperature > ABA > high salinity. 3. To investigate the role under various abiotic stresses, TaSnRK2.8 was transferred to Arabidopsis under control of the CaMV-35S promoter. Overexpression of TaSnRK2.8 resulted in enhanced tolerance to drought, salt and cold stresses, further confirmed by stronger roots and various physiological characteristics, including lower osmotic potential, higher relative water content, strengthened cell membrane stability, increased chlorophyll content and enhanced PSII activity. Meanwhile, significantly decreased total soluble sugar was detected in TaSnRK2.8 overexpressing plants, suggesting that TaSnRK2.8 might be involved in carbohydrate metabolism. Moreover, the expression levels of ABA biosynthesis (ABA1, ABA2), ABA signaling (ABI3, ABI4, ABI5), stress-responsive genes, including two ABA-dependent genes (RD20A, RD29B) and three ABA-independent genes (CBF1, CBF2, CBF3), were generally higher in TaSnRK2.8 plants than in WT/GFP control plants.4. To address the subcellular localization of TaSnRK2.8 in living cells, a construct containing TaSnRK2.8 fused in-frame with GFP driven by the CaMV 35S promoter was expressed in living onion epidermal cells and Arabidodsis. Both results showed the presence of TaSnRK2.8 in the cell membrane, cytoplasm and nucleus.5. TaSnRK2.8 in genome was about 6.1 kb, which contained 9 extrons and 8 introns. Sequencing results showed that two types of sequences were detected from common wheat, named TaSnRK2.8-A and TaSnRK2.7-B. TaSnRK2.8-A were mapped on chromosome 5A with nullisomic lines of Chinese Spring. One hundred and sixty five common wheat accessions were used to detect SNP of TaSnRK2.8-A-C in genomic sequence through sequencing. Three SNPs were detected in 165 TaSnRK2.8-A-C sequences. All the SNPs belong to transitons, and the frequency of SNP was 1 SNP/250 bp. There was no insertion/deletion. The nucleotide diversity (π), i.e., the average pairwise sequence differences between two random sequences in a sample, was 0.00068 per site. The average estimate ofθw, which is based on the observed number of polymorphic sites in a sample, was 0.00070 per site. The nucleotide diversity value in the 3′UTR was highest, lower in intron, the lowest in extron. Neutrality test indicated that there was no selection in TaSnRK2.8-A-C region. Association analysis of traits between A and G genotype (5917 bp, A→G) indicated that TaSnRK2.8 was involved in water-soluble carbohydrate, plant height, flag leaf width, seedling biomass, heading stage and drought resistance index. The A-allele was a preponderant allele in the 165 wheat varieties.6. In order to analyze transcriptional regulative mechanism of TaSnRK2.8 gene, the TaSnRK2.8 promoter (1797 bp) was isolated from wheat. The sequence is abundant in A/T base and predicted to contain a lot of putative cis-elements, such as TATA-box, CAAT-box, ABRE, HSE, TC-rich repeats, LTR and C-repeat/DRE. To identify the key promoter regulative region controlling gene expression, TaSnRK2.8 promoter was truncated according to those putative cis-elements, and inserted into the site upstream of GUS reporter gene. Then, vectors containing different length TaSnRK2.8 promoters were transferred into Arabidopsis. Histochemistry staining analysis showed that only the longest promoter fragment (Dp1870) could activate the expression of GUS gene under normal conditions. Meanwhile, GUS was only expressed in petioles and stems, while not observable in leaves and roots. GUS fluorescence intensity analysis indicated that GUS activity under control of the Dp1870 promoter was lower (0.16~0.35 times) than that under control of the CaMV-35S promoter.In summary, TaSnRK2.8 is a multifunctional regulatory factor, which could participate in sugar metabolic and stress signaling in wheat. It may be possible to utilize TaSnRK2.8 in the improvement of abiotic-stress tolerance in crop species.更多还原