【作者】 李翠玲；

【导师】 夏光敏；

【作者基本信息】 山东大学 ， 细胞生物学， 2008， 博士

【摘要】 植物的耐盐性是由多基因控制的复杂的数量性状,盐胁迫应答相关的基因涉及到代谢、细胞防御、能量、离子平衡和转运、细胞生长和分裂等诸多方面,所有这些基因构成一个复杂的调控网络。因此,对盐渍胁迫下植物基因表达整体概况的研究有助于我们更好的理解植物的耐盐机制。耐盐小麦品种山融3号是小麦品种济南177（Triticum aestivum L.2n=42）与其小麦族偃麦草属禾草长穗偃麦草（Thinopyrum ponticum 2n=70）经过不对称体细胞杂交技术获得的体细胞杂种渐渗系。遗传及生理生化的分析表明,该品种含有长穗偃麦草染色体小片段,耐盐指数及各项生理生化指标均其耐盐性明显优于亲本小麦。山融3号不同于其它耐盐相关研究所利用的单一遗传背景的材料,其耐盐性由主效基因和微效基因共同控制。本文利用小麦全基因组芯片,研究了山融3号及其亲本济南177经不同时间盐胁迫后不同组织的诱导表达谱,从转录组的水平对小麦盐胁迫诱导基因表达情况进行分析,并从山融3中克隆了3个耐盐相关基因,进行了功能研究,主要研究内容和结果包括:1.利用Affymetrix小麦基因芯片分析小麦盐胁迫诱导的基因表达谱采用Affymetrix小麦全基因组芯片（GeneChip Wheat Genome Array）,首次构建了小麦体细胞杂交耐盐品种山融3号及其亲本济南177盐胁迫诱导的表达谱,一共获得6504个差异表达探针,代表了5,577个盐胁迫诱导的差异表达基因。主要参与主要包括胁迫响应、钙离子结合、转录调控、氮代谢、氨基酸代谢以及氧化还原等过程。在盐胁迫下根系中的差异表达基因比叶片中的大约多一倍,由于根系直接面对盐胁迫,所以其差异表达基因多与离子吸收有关,主要包括许多水通道蛋白、钙依赖的钾离子通道以及一些非选择性通道蛋白等。对盐胁迫特异性诱导或者抑制表达探针的分析表明液泡膜钠氢逆向转运蛋白基因Na⁺/H⁺ antiporter（NHX1）、液泡膜焦磷酸酶基因PPase和TaHKT1在山融3号和济南177中差异表达,揭示离子吸收和区隔化的调控可能在山融3号的耐盐中起了重要作用。部分重要盐胁迫响应相关基因在山融3号和济南177之间也存在差异表达,ABA合成途径相关基因以及ABA响应的因子,JA以及GA信号途径的有关基因在盐胁迫后均上调表达,表明在小麦中激素途径与逆境响应途径也有交叉。组蛋白可以通过转录后修饰来诱导或者抑制许多功能基因的表达,它们在盐胁迫后的大量下调表达暗示其在山融3号的盐胁迫应答中起作用,值得我们进一步研究。2.小麦耐盐相关基因TaCHP的全长cDNA克隆及初步的功能分析对一个功能未知的锌指蛋白基因TaCHP进行了克隆和测序,表达谱分析发现盐胁迫后TaCHP在根系中特异性表达,而且在山融3号和济南177间有明显的差异表达。TaCHP表达不受干旱胁迫、氧化胁迫的影响,而ABA胁迫后其表达谱与盐胁迫的表达一致,推测TaCHP基因在山融3号的表达与渗透胁迫和氧化胁迫无关,可能是盐胁迫和ABA响应途径共同的调控因子。组织原位杂交结果表明TaCHP在山融3号根系成熟区的皮层细胞和导管细胞中都有表达,说明TaCHP基因参与了山融3号的盐胁迫过程。TaCHP的蛋白结构分析发现TaCHP的氨基酸序列包含三个植物特有的锌指蛋白结构域—DC1 domain,推测其可能参与了植物磷酸肌醇参与逆境胁迫的响应途径。拟南芥中过量表达TaCHP基因后,转基因植株的抗盐性生长明显好于对照,说明外源基因的插入和过量表达可能提高了转基因株系对于Na⁺的耐受性。3.小麦中耐盐相关的蛋白酶抑制剂基因TaWRSI5的分析从山融3号和其亲本中克隆了TaWSRI5基因。该基因在DDRT和基因芯片检测中都受到盐胁迫诱导表达。TaWSRI5编码一个小麦中的BBI型蛋白酶抑制剂（Bowman-Birktype Protease Inhibitor）,具有胰蛋白酶抑制活性。表达谱分析表明TaWRSI5基因在H₂O₂胁迫后1小时即被强烈诱导表达,而在盐处理后大约6小时,铝胁迫和干旱胁迫后大约24小时,TaWRSI5基因的表达才到达峰值,所以推测TaWRSI5基因的表达受到H₂O₂的调控,可能参与了多种胁迫响应途径。TaWRSI5的组织原位杂交发现其在山融3号根尖成熟区的内皮层细胞中特异表达,而在济南177的相同位置上没有检测到明显的杂交信号,过表达TaWRSI5基因的拟南芥株系也可以在一定程度上提高转基因株系的耐盐性。揭示TaWRSI5基因可能通过参与调控Na⁺的运输或者调节Na⁺在植物体内的分布来提高植物的耐盐性。小麦耐盐相关基因TaSKC1的克隆及初步功能分析小麦耐盐的一个重要机制是调节离子的吸收,运输从而维持植物体内较低的Na⁺浓度。利用RACE技术分别从山融3号,济南177中克隆了小麦中的同源基因TaSKC1,系统进化分析表明TaSKC1的氨基酸序列与OsSKC1和AtHKT1的同源性最高,推测其可能参与Na⁺通过韧皮部汁液从地上部分至根部的再循环过程,从而减少叶片中Na⁺的积累。酵母突变体功能互补试验揭示TaSKC1能够介导盐胁迫后Na⁺的吸收,过表达TaSKC1的酵母细胞对Na⁺更敏感。推测盐胁迫后TaSKC1的表达量降低可能是离子胁迫反馈调节的结果,盐胁迫下植物中由于积累过多的Na⁺,使细胞受到盐害,激活大量盐胁迫响应因子,通过逆境胁迫的信号转导系统,反馈抑制TaSKC1的表达,从而降低Na⁺进入植物体或者向地上部位运输的速率,维持植物细胞内的离子平衡。4.小麦体细胞杂交与基因突变和基因表达差异TaSKC1和TaCHP基因的序列比较分析表明其是来自其小麦亲本济南177,而且在进化过程中非常保守,所以他们在盐胁迫下山融3号和济南177间的差异表达很可能取决于上游调控序列的不同。对TaWSRI5基因序列的分析显示:山融3号中TaWSRI5基因可能来自长穗偃麦草与济南177中的同源基因重组,另外有少数碱基发生了点突变。研究结果从分子水平上验证了体细胞杂交可以导致受体的基因组的剧烈变化,不仅可引起受体功能基因的结构变化,而且还导致其表达水平的变化,这种的变化可促进新基因、新性状的形成和小麦种质创新。总之,在转录水平上对山融3号和济南177的研究发现体细胞杂交导致了二者基因组中大量基因的差异表达,其中山融3号中特异诱导或者抑制表达的基因可能是影响其耐盐性的重要相关基因。对山融3号中的TaCHP、TaWRSI5和TaSKC1基因的研究表明,它们可能是山融3号复杂的耐盐调控网络的重要组分。更多还原

【Abstract】 As an important agronomic trait in crop plants, salt stress tolerance is controlled by quantitative trait loci （QTLs）. Salt stress condition may activate multiple signaling pathways, and enhance or inhibit downstream effect genes, including genes related with metabolism, defense, ion influx and transport, reestablishing homeostasis, biosynthesis of osmoprotectants and some cellular structures, etc. All of these genes make full functions coordinately to keep plant growing and developing regularly. Therfore, it is necessary to analysis the whole genome transcriptional profile of plant under salinity stress.A new somatic hybrid introgression line Shanrong No.3 （hereafter SR3） has been generated in our lab from hybridization of common wheat Jinan 177 with Thinopyrum ponticum, a salt and drought tolerant grass. Cytological and molecular analysis showed that some nuclear and non-nuclear DNAs and even functional genes of donor Th. ponticum were introgressed into this line. SR3 had a significantly higher yield than its parent JN177 and the salt-tolerant control cultivar in salt- alkali soil of Shandong, China. It has passed Shandong provincial regional yield trial for new salt-enduring wheat cultivar （Lu-Nong-Shen-Zi No. [2004]030）. The result of SSR marker analysis suggests that a major salt tolerance gene and some microgenes controlled the salt tolerance of SR3.In order to investigate the mechanism of SR3 response to salinity, we analyzed genome-wide transcriptional analysis of two genetically related wheat genotypes （SR3 and JN177）. The data come from wheat genome array of different tissues at different times under a gradually imposed salinity stress. Based on the analysis from transcriptional profiles between the SR3 and JN177. as well as by using the RACE method, three full-length cDNA related to salt stress were cloned from SR3 and their expression characters and functions were analyzed. The main research contents and results achieved in this work were summarized as follows. 1. Comparative transcriptional profiling of SR3 and JN177 under salinity stress using the affymetrix wheat genome arrayWe used the Affymetrix wheat genome array containing 61127 probe sets to explore the difference in transcriptome of the wheat salt-tolerant genotypes SR3 and it wheat parent JN177 under control and salinity-stressed conditions during vegetative growth. A totally of 6,504 probe sets, on behalf of 5,577 transcripts were identified with differential expression patterns between SR3 and JN177 under salt stress or no-salt stress conditions. These genes were mainly involved in the process related to material transport, ribosome, membrane, calcium ion related signal transduction, environment stress response, transcript regulation as well as cell wall organization and biosynthesis. Bioinformation data shows that under salt stress, the differential expression probe sets in root are probably two times of that in leaf blade. Because the root of plant faces the salinity stress directly, a lot of the difference expressed genes in root were involved in the ion absorption and transport, mainly include many water channel proteins, the calcium-dependent potassium ion channel and non-selective channel proteins. Salt specific responsive probe sets includes some famous salt tolerance related genes, e.g. Na⁺/H⁺ antiporter gene （NHX1）, PPase and TaHKT1, that was induced or inhibited in SR3. This implies that the sodium absorption and ion compartmentation in vacuole play an important role in the SR3. It was also found that some probe sets showed differential expression between SR3 and JN177 also involved in the transcriptional response to salt stress. Some probe sets responsive to salt stress were further analyzed including: ion transporter. ABA metabolism, signal transduction pathway and responsive genes, histone, proline synthesis and metabolism pathway. Some genes in the hormone signal transduction pathway like ABA, JA and GA were induced after salinity treatment, which suggested that there is a crosstalk between the hormone and abiotic stress. Histone may induce or suppress the expression of many function genes through the post-transcription processing, which were generally suppressed in SR3 after salt stress with a specific pattern and worth for further study.2. Cloning and functional analysis of the full-length cDNA of TaCHP gene involved in salt stress The full-length cDNA of TaCHP was cloned and sequenced from SR3 and JN177. Expression of TaCHP in SR3 is down regulated under salt stress but up-regulated in JN177 based on the array data analysis. RT-PCR result revealed that the transcription of wheat TaCHP gene was suppressed in salt-tolerant line SR3 under saline stress and ABA treatment and showed low-expression level in JN177, but no-affected under drought and H₂O₂ treatment. That implied that TaCHP gene acts as a co-regulator of the salt and ABA response pathway. Analysis of in situ hybridization shows that TaCHP gene expresses in the cortex and vessels of xylem in the maturation region of root of SR3 under control condition, while no signal was detected in the root after 24h salt stress.The deduced amino acid sequence of TaCHP gene showed homologous to CHP-rich zinc finger protein-like of Arabidopsis and rice, with 3 divergent C1 domains that only found in plant proteins. This short domain is rich in cysteines and histidines and probably also binds to two zinc ions. The function of proteins with this domain is uncertain in plant; however this domain may bind to molecules such as diacylglycerol and take part in the PLD/PKC signal transduction pathway. The transgenic Arabidopsis lines over-expressed TaCHP gene exhibited enhanced resistance against salinity stress.3. TaWRSI5, a Bowman-Birk type protease inhibitor, is involved in the tolerance to salt stress in wheatA salt responsive gene TaWRSI5 was characterized from salt tolerant cultivar SR3, which was induced after salt stress in wheat genechip and the silver staining mRNA differential display. The peptide encoded by TaWRSI5 contains a Bowman-Birk domain sharing a high level of sequence identity to monocotyledonous protease inhibitors. When expressed in vitro, the TaWRSI5 gene product exhibited trypsin, but not chymotrypsin inhibition. The expression level of TaWRSI5 was increased in SR3 roots exposed to salt, drought or oxidative stress while with different peak time of H₂O₂ （1h）, salt （6 h）, AlCl₃ （24 h） and PEG （24 h） in roots. That suggested H₂O₂ may act as an upstreaming regulator of TaWRSI5 and take part in abiotic stress. In situ hybridization showed that it is induced in the endodermal cells of the mature region of the SR3 root tip, with no signal detectable in the corresponding region of the salt susceptible cultivar JN177. SR3 has a higher selectivity for K⁺ over Na⁺. and therefore limits the transport of Na⁺ from the root to the shoot. When over-expressed in Arabidopsis thaliana, TaWRSI5 improves the ability of seedlings to grow on a medium containing 150 mM NaCl. We suggest that TaWRSI5 plays an important role in regulating long-distance Na⁺ transport or the sodium distribution in SR3 plants exposed to salt stress.4. Isolation and characterization of the TaSKC1 gene from SR3 involved in salt stressAn important mechanism of salinity resistance in wheat is adjusting the sodium-selective transport from root to shoot and retaining low Na⁺ density in shoots. No candidate gene has been reported in wheat for this process, and there are not any corresponding probe sets on the wheat genechip. Homologous cDNA of OsSKC1 in wheat- TaSKC1 was cloned using the RACE methods. The deduced amino acid sequence exhibits 71% identity to that of OsSKC1 and the next to AtHKT1. To determine the TaSKC1 function, we expressed the gene in Saccharomyces cerecisiae mutant strain G19, which had defect in the Na⁺ efflux system and show Na⁺ sensitive phenotype when grows in medium with NaCl. The result showed that G19 strain with TaSKC1 overexpression displays increased Na⁺ sensitivity phenotype than the control with empty vector. This result suggested that TaSKC1 could mediate Na⁺ uptake in yeast mutant strain G19. The transcription of TaSKC1 gene is down-regulated in both SR3 and JN177 under saline stress, oxidative stress and ABA treatment, with a different degree. The putative mechanism was that: there are massive Na⁺ accumulated in the root cell after salt stress and lots of stress responsive genes were induced, leading to the negative regulate to TaSKC1 gene and suppressing its expression, then reducing the Na⁺ uptake from the roots to shoots so as to keep low Na⁺ content in shoots of wheat.5. Somatic hybridization could promote the mutation in genome sequence and differental expression of geneWe cloned the TaCHP1 and TaSKC1 genes homologous to the parents of SR3, JN177 and Thinopyrum ponticum. Sequence analysis showed that TaCHP1 and TaSKC1 genes of SR3 have the same cDNA sequences with its wheat parent, JN177. It implies that the difference expression among SR3 and JN177 is possibly decided by the different upstream regulation sequence. This result provides a clue to the suggestion that somatic hybridization can affect and regulate the expression pattern of genes in acceptor. As for the third genes, half part of cDNA sequence of TaWRSI5 shows high similarity to that of Thinopyrum ponticum and another half are homologous with that of JN177, with several dot mutations. This implies that TaWRSI5 of SR3 is a new gene derived from the recombination of homolog from SR3 and JN177 in the somatic hybridization by a similar mechanism to the HMW-GS genes that we reported. It gives a molecular proof that somatic hybridization both affects the genome constitution and regulates the expression of some functional genes. This kind of change may promote novel gene formation and enhance wheat germplasm diversity.In summary, the transcriptional profiles of somatic hybrid SR3 shows great difference with its wheat parent JN177 under salinity treatment. This could be the result of the introgression of Thinopyrum ponticum chromatin to wheat in the process of somatic hybridization. Among the different expression genes, some salt specific responsive ones could function in the salinity enhancement of SR3. Of these genes, TaCHP, TaWRSI5 and TaSKC1 studied in this work likely play an important role in the complex network controlling the salt tolerance of wheat variety SR3.更多还原