【作者】 孙雷；

【导师】 熊立仲；

【作者基本信息】 华中农业大学 ， 生物化学与分子生物学， 2017， 硕士

【摘要】 水稻是最重要的粮食作物之一,环境胁迫特别是干旱严重影响了水稻的生长发育,制约水稻产量的提高。植物在自身的进化过程中,形成了许多应对干旱胁迫的机制,其中之一就是表皮蜡质层的形成。本室前期通过大田干旱表型筛选实验,成功从T-DNA突变体库中筛选到一个对干旱抗性降低的突变体dwa1。在干旱胁迫下,dwa1突变体表皮蜡质出现缺失,并伴随体内茉莉酸含量的积累。DWA1编码一个含有2391个氨基酸的巨型蛋白,该蛋白包含5种保守结构域,为了进一步探究各个结构域发挥的生物学功能,本研究将DWA1蛋白以各个保守结构域为单位进行划分,分别构建了单结构域以及多个结构域组合的互补与超表达载体,并转化到了粳稻品种中花11中。同时,对部分结构域的重组蛋白进行了体外原核表达和纯化,为后续对单结构域的功能研究如酶活测定、结构生物学等实验做准备。为了寻找调控DWA1的转录因子,我们收集了本室非生物逆境处理的水稻表达谱芯片数据对DWA1进行共表达分析,发现了一个与DWA1共表达的基因OsWR1(LOC_Os02g10760)。OsWR1与拟南芥蜡质合成基因SHN1同源性很高,编码一个包含AP2结构域的转录因子。通过启动子序列分析发现DWA1上游序列含有与OsWR1结合的类GCC-BOX序列以及DRE/CRT序列。对dwa1突变体、dwa1突变体的全长互补和DWA1全长超表达遗传转化材料在苗期干旱胁迫过程中的激素含量测定和基因表达量检测显示,所有材料在干旱早期JA含量出现迅速上升,在胁迫后期ABA含量迅速上升,参与蜡质合成的基因Wda1前期表达量很低,在干旱后期随ABA含量升高表达量出现大幅升高。突变体内参与茉莉酸合成的基因LOX2在早期表达量显著高于野生型并随干旱胁迫降低,突变体内参与蜡质合成的基因Wda1在干旱后期表达量低于野生型,这与突变体在干旱胁迫下表皮蜡质缺失的表型相吻合。为了在全基因组水平上研究DWA1对水稻干旱胁迫抗性的影响,对dwa1突变体,野生型和DWA1全长超表达材料进行苗期干旱胁迫,取胁迫前后叶片样品进行全基因组表达分析。对数据进行质量过滤并匹配到基因组,分析了各个材料在胁迫前后的基因表达变化。研究发现突变体中胁迫前后的差异基因比野生型少。并且和野生型相比,一些JA合成和蜡质合成相关的基因在胁迫下受到显著的调节。对差异基因进行GO富集分析,发现这些基因在脂类合成和运输的途径中有显著富集,表明DWA1通过调控蜡质和JA的合成途径参与了水稻对干旱胁迫的应答。研究表明DWA1在干旱胁迫下同时参与了干旱与表皮蜡质的合成来控制抗旱性,对DWA1研究的深入对改良农作物品种抗旱性有重要价值。更多还原

【Abstract】 Rice is one of the most important staple crops in China.The yield is being largely decreased due to various environmental stresses,especially drought which has already become the major limiting factor.In order to adapt to drought stress,rice has developed many biochemical and metabolic mechanisms.One of the typical features is the cuticular wax preventing plants from suffering negative stresses.Previously in our lab we isolated a gene from T-DNA insertion mutants and named it dwa1.The mutant is sensitive to drought treatment.Under drought stress in dwa1 mutant,the cuticular wax is severely impaired while the content of JA remains higher than the wild type.DWA1 encodes a mega protein that has 2391 amino acids which has five conserved domains.We divided the whole protein by domains,then we constructed several vectors for genetic transformation including different domain combinations and transformed them into japonica cv Zhonghua11.Also,protein expression experiment was conducted.Each single domain was successfully induced and purified in vitro.This preparation may help further functional study of DWA1,e.g.,enzyme activity,structural biology.In order to find the transcription factors that regulate DWA1,We collected the microarray data from our previous work in our lab that is related to abiotic stress.After the co-expression analysis with DWA1,we found a gene OsWR1(LOC_Os02g10760)that is homologous to SHN1.SHN1 encodes a transcript factor that has something to do with wax synthesis in Arabidopsis.Both OsWR1 and SHN1 contain an AP2 domain.The binding sites of Os WR1,GCC-BOX-like and DRE/CRT-like sequences,were found in the upstream region of DWA1 by promoter analysis.We sampled the seedlings of dwa1 and its genetic transformation lines together with wild type under drought condition and detected the contents of hormones and gene expressions.The result indicates that the content of JA raised in all lines in the early drought stage,while ABA raised in the relatively late drought stage.The expression of Wda1,a gene which is related to wax synthesis,was low at the beginning and then increased when drought stress continued along with the rise of ABA.Compared with the wild-type,the expression of LOX2,a gene which is involved in JA synthesis pathway,was higher in the mutant lines and decreased when drought treatment continued.The expression of Wda1 is lower in the mutant than the wild-type in the late stage.This result is consistent with the wax-absent phenotype in mutant under drought conditions.To study the function of DWA1 on drought resistance,a genome-wide expression profiling for seedlings of dwa1 and its genetic transformation lines was conducted.After the procedure of quality filter and genome match,we analyzed the gene expression changes in different lines.The result shows dwa1 has fewer differentially expressed genes than the wild type under drought conditions.Genes involved in JA and wax synthesis are significantly modulated in dwa1 mutants under drought conditions.Gene Ontology analysis shows the differential expressed genes are involved in lipid metabolism and transportation which indicates that DWA1 takes part in drought stress response by regulating wax and JA synthesis.The results suggest DWA1 controls drought resistance by regulating both JA and wax synthesis.A deeper research of DWA1 may have significant implications for improving the drought-resistant varieties.更多还原