【作者】 吕建；

【导师】 夏光敏；

【作者基本信息】 山东大学 ， 遗传学， 2010， 博士

【摘要】 小麦的耐逆性是由多基因控制的复杂形状,耐逆性的提高需要胁迫应答、离子转运、次生代谢以及能量代谢等多方面因素。目前分子生物学研究及应用已经显著提高了水稻、玉米等对不良环境的抵抗性,但在小麦中,这种进展相对比较缓慢。因此有必要加强加深对小麦逆境下分子机理的研究,而现阶段主要的方式是从转录组变化中找出候选基因,进而验证基因在抗逆中的功能,已经有一些成功的报道。但对于普通小麦这样一个异源六倍体来讲,对其耐逆基因的报道很少,对其整个耐逆响应网络还缺乏研究。小麦渐渗系山融3号(SR3),是普通小麦济南177(Triticum aestivum L.2n=42)同耐盐小麦近缘属长穗偃麦草(Ae)(Thinopyrum ponticum 2n=70)不对称体细胞融合后代。SR3耐逆性高于JN177,特别是在盐/旱胁迫下。SR3同JN177的对比研究,提供了一个很好的研究小麦耐逆机制的平台。早期的研究表明,SR3基因组中渐渗了Ae的基因组序列,其抗逆性是由一个主效基因位点和多个微效基因控制。但具体到哪些基因在起作用,还不是很清楚。在实验室前期对SR3重要耐逆相关基因TaCHP和TaCEO的功能研究中,发现它们在过表达拟南芥中增加抗逆性的同时,都影响AtMYB15的表达。而在拟南芥中,MYB家族是最大的一个转录因子家族；其中一些MYB基因可以显著提高拟南芥抗逆性。类似的在水稻和玉米中,一些MYB基因参与到抗逆响应中。而对小麦MYB家族在抗逆性中作用的研究还没有报道。本论文通过分析SR3芯片数据,整理出了38个响应胁迫的MYB基因(Unigene/EST)。通过RT-PCR分析,系统研究了小麦渐渗系MYB基因对盐/旱胁迫的响应,揭示MYB基因参与耐逆调控的可能方式,并分析了MYB基因发育周期中的表达模式；对JN177/SR3间MYB基因等位变异进行了分析,进一步阐明MYB参与胁迫的方式及内容；选择了其中一个在JN177与SR3的具有序列差异的Tamyb31基因,进行功能鉴定。主要的研究内容及结果包括：1.MYB基因信息的整理在实验室芯片数据中,通过探针序列的BlastN分析,得到了SR3和JN177三叶期表达的36个MYB基因对应的Unigene(EST)信息。由于MYB家族在水稻和拟南芥中较为庞大,分析的这36个MYB基因只是小麦MYB基因家族的一部分。为此,又进一步从山融3号cDNA文库中克隆到了另外两个MYB基因,加起来总共有38个基因。2.MYB基因对盐/旱胁迫的响应分析及发育周期表达模式分析通过RT-PCR方法研究了SR3及JN177在盐/旱胁迫下不同时间点的差异,验证了芯片结果。同时发现在6个时间点间,35个MYB基因响应盐/旱胁迫一共存在42种表达模式(另外3个MYB低于RT-PCR的检测限)。不同MYB基因在同样的处理下,有着不同的响应模式,例如盐处理SR3叶中MYB3/17呈L1型表达模式,快速响应盐胁迫处理,在0.5h达到峰值,之后持续降低表达量；而MYB 1/8/9/11/15/16/19/27/31/33则呈L6型表达模式,即呈现波浪形表达模式,在0.5/6h到峰值。这表明在同样的器官同样的品种同样的处理下,各个MYB之间对胁迫信号的响应不同,这可能与MYB基因的功能差异有关。同一MYB基因在不同胁迫下的响应模式不一致,例如SR3叶中,MYB31在盐/旱胁迫下表达模式分别为L6/L41型。L6型呈现波浪形变化；L41型呈现一种逐步下降的情况,在0.5h升至最高,之后表达逐渐下降,在12h略有回升,之后接着下降。这表明MYB31在盐/旱胁迫信号转导中的位置不同,也就是体现了盐/旱胁迫信号转导的差异。同一MYB基因在不同的器官中表达模式不一致,例如盐处理下SR3中的MYB4,在叶/根中分别呈L11/L16型表达模式,L11型在3h有个表达高峰,而在其余时间点表达量变化不明显；L16型则是从开始处理后就呈现下降趋势。这体现了盐胁迫下,根和叶的同一MYB基因对胁迫的响应不同。同一MYB基因在SR3和JN177中间的表达模式也不一致,在盐处理下的根中,MYB3分别呈L15/L7型表达模式。L15型是Oh之后表达量就开始下降,L7型则是呈波浪型变化。这种差异体现了不同品种对胁迫响应的差异,可能是造成对胁迫抗性差异的原因。也有少数几个MYB基因在特定情况下存在一致的表达模式,例如盐处理下的叶中,SR3和JN177中的MYB29都呈L7型表达模式；盐处理下济南177中的MYB15,在根和叶中都呈现出L6型表达模式；JN177根中的MYB13在盐旱胁迫下表达模式都呈现出L7型。整体上看,MYB基因对胁迫的响应比较复杂。如何去判断哪些MYB基因及其表达模式同SR3/JN177抗逆性差异相关。我们从两方面入手：第一方面,找出SR3/JN177各自特异的表达模式。在42种表达模式中,SR3/JN177间差异的表达模式有13种。SR3中特异的响应模式有L3/L1 1/12/13/20/23/37/38型,而JN177中特异的有L28/29/30/34/35/36型。这些差异对我们选择候选基因提供了线索；第二个方面我们选择单个目标基因进行功能研究,例如,我们鉴定了MYB31对应基因的功能,而其胁迫响应的模式有L6/L7/L17/L18/L26/L41型,这些类型对应的基因是我们进一步研究的基础。由于芯片取材选择了小麦的三叶期；为避免发育时期的限制,进一步比较了SR3不同发育时期MYB基因的表达谱。以上发现有助于提供候选MYB基因用于功能验证,并在育种中加以应用。3.MYB基因结构分析由于小麦含有A,B,D三个基因组,而SR3又是体细胞杂种后代,每个MYB基因在SR3中的拷贝数不清楚；参与胁迫响应的MYB,是不是其RT-PCR产物就仅对应一个转录本?而转录本的差异也会对SR3和济南177抗逆性差异产生影响,特别是当RT结果显示为同一表达模式时。为此我们将RT-PCR产物进行测序,同时又对对应的基因组序列进行了比较。在分析的15个MYB基因中,RT产物中仅有MYB5/22是一个转录本；而其余13个MYB基因,则有多余一个的转录本。这些转录本在JN177和SR3中普遍存在差异,可能是JN177和SR3抗逆性差异的原因之一。具体细化分析时,用等位变异来描述这些差异；我们发现这些差异主要是SNP差异,多数是Substitution, Indel位点仅存在两处。在同一个MYB基因中,不同等位基因位点发生的变化不同,例如在MYB5的两个等位位点,两个位点都存在G/A的SNP多态性,但存在的位置不同。不同的MYB基因,等位变化也往往不同,例如MYB19中,三个等位基因位点都发生了变化；而对MYB18来讲,两个等位基因位点上都没有发生序列上的变化。内含子中SNP-Indel的频率要高于外显子中的频率,而SNP-Substitution的频率在内含子中要低于外显子中的频率。在MYB9/22中,启动子的核苷酸多态性要大于基因编码区域的核苷酸多态性。SNP中的核苷酸转化方式以转换为主。通过分析,同时发现等位基因上的SNP可以使编码的氨基酸发生变化。我们认为在小麦抗逆研究中,除了要关注MYB基因表达模式变化,还要关注同样表达模式下哪些MYB转录本在其中起作用。4. Tamyb31功能研究在以上小麦MYB基因家族中,SR3的MYB31随胁迫上调表达,并且上调比较明显。从cDNA文库中分离得到了其全长,通过同源比对,SR3与JN177中的MYB31存在SNP-Substitution,并且导致其蛋白质氨基酸的变异,将其命名为Tamyb31。Tamyb31 cDNA由192bp的5’UTR,765bp的开放阅读框(ORF)以及298bp的3’UTR构成。通过体外DNA结合试验以及转录活性试验,证实了Tamyb31是起作用的转录本。通过设计特异的引物研究其胁迫响应模式,在山融3号的根中,其表达模式类似MYB31的情况,但在叶中不同于MYB31的表达模式。在盐旱处理下,Tamyb31都是在0.5h就明显上调表达,这表明Tamyb31参与早期盐旱胁迫信号的转导。在激素处理下,Tamyb31在0.5h瞬时上调表达,表明Tamyb31参与多种激素信号的转导。从发育时期来讲,Tamyb31在整个发育期中都有表达,而在拔节期中的茎中表达量最高。Tamyb31编码一个R2R3型MYB蛋白；同其它物种中的MYB蛋白相比,其SANT domain的保守性很高；亚细胞定位于细胞核内；结合Ⅰ/Ⅱ/ⅡG型MYB结合基序。Tamyb31的拟南芥过表达系明显增加了对盐/旱胁迫的抗性。在NaCl处理下,过表达系的根长明显长于对照的根长；同时过表达系增加了对LiCl/KCl的抗性。这表明Tamyb31可能增加对离子胁迫的抗性。在甘露醇处理下,过表达系同对照系的表型没有明显差异。在控水干旱处理时,过表达系存活率要高于对照系；这可能同过表达系较低的失水率有关。Tamyb31也增加了过表达系萌发时对NaCl和ABA的抗性。NaCl处理下,过表达系中AtP5CS1/CBF3基因的表达量高于对照；不处理情况下,AtABF3的表达量在过表达系中较高。Tamyb31可能通过调控这些基因的变化来提高拟南芥的抗性。同时我们又分析了AtCBF3/ABF3的启动子区域,发现两者启动子上有多个MYB结合位点,体外实验也证实Tamyb31可以结合这些启动子区域。Tamyb31与AtMYB15有很高的同源性,但两者在功能上有所差异。例如AtMYB15可以同AtICE1相互作用,而Tamyb31则不可；Tamyb31没有选择性的结合Ⅰ/Ⅱ/ⅡG型结合基序,而AtMYB15则优先结合Ⅱ/ⅡG基序。这表明两者之间在胁迫中的作用有所差异。通过对Tamyb31的研究,我们认为本文的研究方法是有效的,即能分离到在胁迫中起作用的MYB基因。鉴于水稻/拟南芥中多个MYB基因参与胁迫抗性的提高,小麦MYB抗逆相关基因的挖掘仍需继续。更多还原

【Abstract】 Wheat is one of the major crops, so its yield and quality are always quite important social issues around the world, especially in China. Yield and quality of wheat are influenced by varieties of environmental conditions, such as drought and salinity. Therefore, enhancement of wheat stress tolerance is critical for its production to cope with the still-increasing adverse environmental situations allover the world. Stress tolerance is a multigenes-controlled phenotype, which may include stress response, ion transport, secondary metabolism and energy flow. Until now, many molecular results regarding stress tolerance mechanisms were achieved using model plants, and have been applied in rice and maize breeding. However, given its hexaploidy and mega-genome, such progress in wheat is very slow, and only a few genes have been reported to improve wheat stress tolerance, which can not reveal the whole truth of its stress tolerance, and are inadequate for wheat breading.Alternatively, Thinopyrum ponticum, the wild relative of wheat with high stress tolerance, is an excellent gene reservoir for wheat breeding. Via somatic hybridization, we bred a wheat introgression line stress tolerance cultivar Shanrong No.3 (SR3) between common wheat stress sensitive cultivar Jinan 177 (JN177) and T. ponticum. Former results showed that some chromatin fragments of T. ponticum intergrated into SR3’s genome, and a high frequency of allelic variation and a vast transcriptomic and proteomic change in SR3 happened. These alternations may offer the higher stress tolerance of SR3.Previous study in stress responsible genes TaCHP/TaCEO from SR3 indicated that their ectopic expression increased stress tolerance of Arabidopsis, and influenced the transcription of AtMYB15 in transgenic seedlings. AtMYB15 is one of MYB family members that can improve stress tolerance in Arabidopsis, suggesting MYB family may also play important roles in stress tolerance of wheat. However, little information of MYBs is available in wheat.In this paper, we proposed to identify wheat MYBs based on microarray data of SR3, to analyze their expression pattern under salinity/drought stress and during the whole developmental course, and to compare the variation in their cDNA and genomic sequences between JN177 and SR3, with the aim to primarily characterize the relationship between MYB family and stress tolerance of SR3 and find golden MYB candidates for wheat breeding. The main research contents and results of this work are summarized as follows.1. Wheat MYBs identification from SR3Based on microarray data in our lab,36 MYB unigenes/ESTs were identified using BlastN analysis. The other two MYBs were identified from the SR3 cDNA library in our lab. Totally,38 MYBs with detectable transcription patterns in SR3 at three-leaf stage were identified for further study.2. Expression patterns of MYBs under stressUsing RT-PCR, the express characteristics of 35 MYBs under salinity/drought stress and during the whole development course were analyzed and classified into 42 patterns s in total(3 MYBs were below detectable limit of RT-PCR).MYBs showed differential patterns in response to the same stress. For example, in salinity-stressed SR3 leaves, MYB3/17 showed L1 pattern, a transient response to salinity; while MYB 1/8/9/11/15/16/19/27/31/33 showed L6 pattern, with fluctuant transcription during stress. This indicated that different MYBs play different roles in salinity response.MYBs took different patterns under salinity from those under drought. For instance, in SR3 leaves, MYB31 took L6 pattern under salinity stress, and L41 pattern under drought stress. L41 pattern had a high and a low transcription peak at 0.5h and 12h timepoint, respectively. This indicated that MYB31 plays different roles in salinity and drought response.MYBs showed organ-specific expression patterns. In salinity-stressed SR3, MYB4 showed L11 and L16 patterns in leave and root respectively. L11 pattern is that only mRNA abundance at 3h timepoint was higher than the control. L16 pattern is the transcription that was gradually reduced during stress. This indicated stress response signal transduction chains carry out in an organ-specific manner, and MYBs may function differently in different organs. MYBs had cultivar-specific expression patterns in response to stress. Under salinity stress, MYB3 showed L15 pattern in JN177 roots, whereas L7 pattern in SR3 roots. L15 and L7 patterns are similar to L16 and L6 patterns, respectively. These differences may account for different stress tolerance ability between JN177 and SR3.Apart from the above categories, a few MYBs also acted in the same way under specific conditions. In total, the diverse expression patterns of MYBs suggesting that this family is involved in stress response in a complex way.In order to rule out the relationship between MYBs and the difference in stress tolerance of SR3 from JN177, two analyses were conducted. One is checking SR3-or JN177-specific expression patterns. Among 42 expression patterns, eight (L3/L11/12/13/20/23/37/38) are SR3-specific, and five are JN177-specific. MYBs showing these cultivar-specific expression patterns should be the candidate genes for further study. The other is based the function study of MYB31 (Tamyb31), whose over-expression can improve stress tolerance. MYBs in the same expression patterns as MYB31 are also candidates.Besides, expression patterns of these MYBs at various stage of developmental course in SR3 were checked. The results displayed that MYBs also had their individual expression characteristics during development.3. Sequence variation of MYBsCommon wheat is hexaploid, and SR3 is a somatic hybrid of common wheat. This implies that the RT-PCR product of a certain MYB gene of wheat, especially SR3, may contain more than one transcript. If it is the case, these different transcripts may play different roles in stress response.In order to outline this question, the RT-PCR products and genomic sequence of 15 MYBs were further cloned. Firstly, except for MYB5/22, the other 13 MYBs had more than one transcript in the RT-PCR products. The difference between transcripts concluded two types of diversity sites:Substitution and InDel. The frequency of Substitution was higher than that of InDel. Secondly, distribution of SNP-Indel varied in different MYBs, different alleles. In introns, frequency of SNP-Indel was higher than that in exons; while frequency of SNP-Substitution was a little lower than that in exons. In MYB9/22, Pi value in promoter region was higher than that in coding region. As for Substitutions, frequency of conversion was higher than that of transversion.Above results indicates that SR3 genome suffered a high-frequency of variation. Such sequence variation between JN177 and SR3 may result from two ways, one is the introgression of T. ponticum chromatic fragments, and the other is the genomic shock during somatic hybrid process. SNPs between JN177 and SR3 could cause change in amino acids, so it is quite important to check which transcripts function in stress response in wheat.4. Functional analysis of Tamyb31TaMYB31 was selected as an important candidate gene because of its significant upregulation of transcription under stress in both SR3 and JN177. Its full length cDNA was isolated from cDNA library of SR3, consisting a 192-bp 5’UTR, a 765-bp ORF and a 298-bp 3’UTR. There are SNPs in SR3 and JN177, which led to amino acide variation of the TaMYB31, so TaMYB31 of SR3 was named Tamyb31. Using DNA binding assay and transcriptional activity assay, the transcript with MYB function was identified.RT-PCR and real-time PCR analysis showed that Tamyb31 was induced after 0.5h exposure to salinity/drought stress in SR3. Under treatment with various plant hormones, the expression of Tamyb31 peaked at 0.5h, indicating Tamyb31 may be involved in many signal pathways. Tamyb31 expressed at the whole developmental course, with the highest level at stem jointing stage. Tamyb31 encodes a R2R3 MYB protein with conserved SANT domain when compared with other MYBs; Tamyb31 localizes in nuclei, and binds toⅠ/Ⅱ/ⅡG MYB binding motif.Overexpression Tamyb31 in Arabidopsis increased the tolerance to abiotic stress. Under NaCl, LiCl and KC1 stress, roots of transgenic lines were significantly longer than those of the control, indicating that Tamyb31 can increase the ion stress tolerance of plants. Under drought stress, the overexpression lines also grew better than the control lines. Under mannitol treatment, however, no obvious difference was detected between the overexpression lines and the control lines. This suggests that the enhancement in drought tolerance may be achieved through lowing water loss rate. Under salinity stress, AtP5CS1/AtCBF3 had higher transcripts in the overexpression lines than in the control lines; under non-stressful conditions, AtABF3 had more mRNA abundance in the overexpression lines than in the control lines. Tamyb31 can bind promoter regions of AtABF3/CBF3 using yeast one hybrid assay. These findings suggested that Tamyb31 may confer with abiotic stress through affecting the transcription of these genes.Tamyb31 shared high amino acid sequence similarity with AtMYB15, but their action mechanism may be significantly different from each other. For example, Tamyb31 did not interact with AtICE1, while AtMYB15 did. Tamyb31 had equal binding ability to I/II/IIG MYB motifs, while AtMYB15 prefers to II/IIG motifs.Taken together, in the case of Tamyb31, we conclude that gene-family-ome analysis is a feasible strategy for key functional gene isolation with the aim to stress response mechanism elucidation and transgenic engineer assistant breeding. Besides Tamyb31, other identified MYBs are also worthy of further study.更多还原