【作者】 邹锡玲；

【导师】 郑用琏；

【作者基本信息】 华中农业大学 ， 生物化学与分子生物学， 2011， 博士

【摘要】 渍害和干旱、盐渍、异常温度共同影响着全球的物种分布和产量。玉米是世界上最重要的粮食作物之一,在我国由于季节性降雨尤其是在长江中下游地区,渍害已经成为玉米高产、稳产以及扩大种植面积的重要制约因子。本研究旨采用反向遗传学的研究策略,以玉米耐渍自交系HZ32为材料,筛选玉米苗期根系在淹水胁迫后期过程中诱导表达的基因,揭示玉米苗期在淹水胁迫后期获得适应性应答的分子机制,克隆重要的调控基因,并在进行功能分析的基础上开发可利用于耐渍性改良的分子标记辅助选择的功能标记。取得的主要研究结果如下：1.以耐渍自交系HZ32为材料,选取淹水处理后四个时间点(12h、16h、20h以及24h)的幼苗根系构建正向抑制消减文库,以反向Northern为手段对该文库的768个克隆进行筛选,共筛选到465个诱导表达的EST,共代表295个unigene基因,涉及到了一系列功能,例如信号传导、蛋白质降解、离子运输、碳氮代谢以及转录和翻译调控类基因。表达分析表明,氨基酸代谢链接了蛋白质降解和碳代谢途径,在淹水胁迫后期起着重要作用,并且可能参与了细胞质pH调节以及降解氨基酸碳架给糖酵解途径提供中间产物以支持能量供给。与淹水胁迫前期比较,低氧信号传导途径在淹水后期依然十分活跃,区别于前期,可能参与调节植物后期对长期淹水胁迫的适应。推测淹水胁迫下玉米幼苗根系的应答过程大致可以分为2个阶段：防御和适应。本实验室筛选到的信号传导相关基因可能调控植物在长期的淹水胁迫下进行有效的适应型的应答反应。通过电子定位策略,筛选到的其中63个诱导表达基因与前人定位到的耐渍QTL区间共定位,这些基因可能是重要的候选基因,但仍需进一步验证。2.在上述消减文库中筛选到一个编码脯氨酰羟化酶的基因zmP4H,该基因在淹水胁迫后期在HZ32苗期根系中呈现上调表达。该基因在动物中已被证实是在低氧胁迫下信号感受和传导的关键基因,然而该基因在玉米乃至植物中耐渍应答过程中的作用尚未明了。利用生物信息学方法对玉米全基因组zmP4H的结构、保守motif、系统进化关系以及其在染色体上的区段进行了分析。本研究中分离得到9个编码脯氨酰羟化酶的基因,命名为zmP4H1-9。研究发现其中的5个基因涉及到选择性剪接,这5个成员通过选择性剪接至少产生了19个转录本。同一个基因在不同的自交系中发生的选择性剪接方式不尽相同,这可能与选择性剪接的时空特异性和逆境应答特异性相关。对这些选择性剪接的转录本进行分析,结果表明剪接位点的选择与剪接位点附近的碱基组成密切相关,而那些非组成型的剪接位点的选择很可能与位于剪接位点处的碱基重复序列相关。11个选择性剪接转录本均含有提前终止的密码子,这使得这些转录本很可能遭到无义介导的RNA降解系统的降解。在耐渍材料HZ32中,采用RT-PCR表明zmP4H因在淹水胁迫下表现出不同的应答反应。而选择性剪接产生的转录本在淹水胁迫下也表现出不同的丰度变化,这表现在同一个基因的不同剪接转录中,而不同基因的选择性剪接转录本也如是。这表明在HZ32中,zmP4H在淹水应答过程中受到选择性剪接这种转录后调控机制的调控。3.以本课题组前期筛选到的耐渍相关EST为基础,利用RACE技术,从玉米耐渍自交系HZ32克隆得到zmbRLZ基因全长,序列分析显示, zmbRLZ蛋白质由282个氨基酸组成,从第118位到第182位肽段与BRLZ5和bZIP高度同源,且含亮氨酸重复序列。real time PCR结果显示zmbRLZ基因的表达量在耐渍材料HZ32中淹水后4h达到最大,而在敏感材料Mo17中其表达量则基本维持不变。构建zmbRLZ与GFP融合载体,洋葱表皮的瞬时表达结果表明,zmbRLZ编码的蛋白定位于细胞核,说明zmbRLZ蛋白是能在细胞核中发挥功能的转录因子。对zmbRLZ利用大肠杆菌系统进行体外蛋白质表达,并利用GST标签纯化该蛋白质,凝胶阻滞实验表明,该融合蛋白能够与Adhl基因启动子中的厌氧应答元件序列发生特异地结合,推测其能够调控下游启动子区域里含有ARE顺式元件的应答基因的转录表达。根据自交系HZ32和K12在zmbRLZ基因序列中的SNP设计引物,成功开发了基于SNP的CAPS标记zmbRC/G,利用已构建的(HZ32×K12)F2群体,将zmbRLZ基因定位在SSR标记umc1743和umc1107之间,该区段与课题组前期通过遗传连锁定位的1个玉米耐渍性QTL的密集区域吻合。更多还原

【Abstract】 Waterlogging, caused by flooding, long periods of rain, and poor drainage, is a serious abiotic stress determining crop productivity worldwide alongside with drought, salt salinity and extreme temperatures. In the middle and lower Yangtze River area, the major corn-growing region of South China, seasonal rainfall greatly affects maize plantation, leading to yield losses. The goal of this study is to identify a serial of genes responsing to waterlogging in the late stage and to reveal the molecular basis of waterlogging tolerance by reverse genetics approach. Furthermore, cloning and characterization of important candidate genes and development of molecular markers based on these genes might provide new strategies to improve waterlogging tolerance of maize through marker assisted selective breeding. The main results achieved are as following:1. In this study, the transcriptome at the late stage of waterlogging was assayed in root cells of the tolerant inbred line HZ32 using suppression subtractive hybridization (SSH). A forward SSH library using RNA populations from four time points (12 h,16 h,20 h and 24 h) after waterlogging treatment was constructed to reveal up-regulated genes, and transcriptional and linkage data was integrated to identify candidate genes for waterlogging tolerance. Reverse Northern analysis of a set of 768 cDNA clones from the SSH library revealed a large number of genes were up-regulated by waterlogging. A total of 465 ESTs were assembled into 296 unigenes. Bioinformatic analysis revealed that the genes were involved in complex pathways, such as signal transduction, protein degradation, ion transport, carbon and amino acid metabolism, and transcriptional and translational regulation, and might play important roles at the late stage of the response to waterlogging. A significant number of unigenes were of unknown function. Approximately 67% of the unigenes could be aligned on the maize genome and 63 of them were co-located within reported QTLs. The late response to waterlogging in maize roots involves a broad spectrum of genes, which are mainly associated with two response processes:defense at the early stage and adaption at the late stage. Signal transduction plays a key role in activating genes related to the tolerance mechanism for survival during prolonged waterlogging. The crosstalk between carbon and amino acid metabolism reveals that amino acid metabolism performs two main roles at the late stage:the regulation of cytoplasmic pH and energy supply through breakdown of the carbon skeleton.2. In the SSH library mentioned above, there was a gene encoding prolyl 4-hydroxylase (P4H) induced in the response to waterlogging in HZ32. In animals, prolyl 4-hydroxylases (P4Hs) are regarded as oxygen sensors under hypoxia stress, but little is known about their role in the response to waterlogging in maize.A comprehensive genome-wide analysis of P4H genes of maize (zmP4Hs) was carried out, including gene structures, phylogeny, protein motifs, chromosomal locations and expression patterns under waterlogging. Nine zmP4H genes were identified in maize, of which five were alternatively spliced into at least 19 transcripts. Different alternative splicing (AS) events were revealed in different inbred lines, even for the same gene, possibly because of organ and developmental specificities or different stresses. The signal strength of splice sites were strongly correlated with selection of donor and receptor sites, and ambiguous junction sites because of small direct repeats at the exon/intron junction frequently resulted in the selection of unconventional splicing sites. Eleven out of 14 transcripts resulting from AS harbored a premature termination codon, rendering them potential candidates for nonsense-mediated RNA degradation. RT-PCR indicated that zmP4H genes displayed different expression patterns under waterlogging. The diverse transcripts generated from AS were expressed at different levels, suggesting that zmP4Hs were under specific control by post-transcriptional regulation under waterlogging stress in HZ32.3. The full length of zmbRLZ was isolated from HZ32 using the technique of rapid amplification of cDNA ends, showed that zmbRLZ was consisted of 282 amino acids and was homogy to the domain of BRLZ5 and bZIP from 118 to 182 amino acids. The expression of zmbRLZ was peaked in HZ32 at 4 h and was not changed in Mo 17 under waterlogging in the roots of seedings. The vector with GFP-zmbRLZ was construeted and transferred into onion epidermis using Agrobacterium, indicating that zmbRLZ was located in nucleus. The encoding sequence of zmbRLZ was cloned into the expression vector and expressed successfully as GST (Glutathione S-Transferase) fusion protein in E. coli. The zmbRLZ-GST fusion protein could bind specifically to the ARE sequence from adhl promoter in vitro by Electrophoretic Mobility Shift Assay, indicating that products of zmbRLZ-GST fusion protein had a function of ARE binding specificity. A marker "zmbRC/G" was developed based on the SNPs of the sequences between HZ32 and K12 and was mapped on the linkage map of a F2 (HZ32×K12) population and was located between the markers umc1743 and umc1107 indicated that zmbRLZ was located in the QTL of waterlogging tolerance.更多还原