【作者】 陈晨；

【导师】 都兴林；

【作者基本信息】 吉林大学 ， 作物学， 2024， 硕士

【摘要】 水稻作为最重要的粮食作物之一,在整个生育期对水分的需求量极大。然而,目前全球的农业可使用水资源是普遍匮乏的。在这种背景下,水稻的生长和发育以及高产稳产受到了严峻的挑战,干旱已经成为其主要的制约因素。因此,挖掘抗旱新基因,深入探究抗旱分子机制,对于水稻的育种研究意义深远。植物WRKY转录因子在植物抗逆过程中起着非常重要的作用。本研究以水稻中的一个转录因子OsWRKY95为研究对象,对其在水稻抗旱性方面的功能进行了探究,主要结果如下:1.对OsWRKY95进行生物信息学分析及单倍型分析,结果表明OsWRKY95属于WRKY转录因子家族Ⅲ类家族成员,OsWRKY95蛋白由260个氨基酸组成,包含一个保守的WRKY域。OsWRKY95基因启动子中含有多个顺式作用元件,其中许多都与植物的逆境胁迫相关。利用3 K水稻SNP数据集对OsWRKY95的单倍型进行分析,结果表明,OsWRKY95有6个单倍型并且在不同的水稻亚种间存在明显的籼粳分化。其中,Hap 1主要分布在粳稻当中,其余几个单倍型则主要分布在籼稻当中。2.为了探究OsWRKY95是否与植物逆境胁迫功能相关,通过荧光定量PCR分析发现OsWRKY95的表达量受多种非生物胁迫和激素处理诱导。OsWRKY95不仅能响应干旱、冷和盐胁迫诱导,而且还受ABA、JA、IAA等激素诱导。在干旱、冷、IAA和ABA处理条件下,OsWRKY95基因在水稻中的表达量表现为先上升后下降。在盐胁迫和JA处理条件下,植物细胞可以有效地抵御外界环境的胁迫,从而维持正常的生理功能。盐胁迫可以抑制植物细胞内膜的渗透性,从而减少膜系统中的水分流失;而JA处理可以抑制植物细胞内膜的渗透性,从而降低细胞内的水分流失。此外,JA处理还可以抑制植物细胞内膜系统中的离子吸收,从而减少离子流失。其表达量在24 h之内持续升高,推测OsWRKY95可能通过ABA、JA信号通路参与调控水稻的生长发育、生物胁迫和非生物胁迫。同时,OsWRKY95基因在水稻中呈组成型表达,即在幼年及成年主要组织中均有表达,并且在穗中表达量最高。3.进一步对OsWRKY95的过表达和敲除植株进行苗期抗旱功能鉴定,在用20%的PEG6000模拟干旱胁迫再使用木村B营养液恢复以后,对幼苗的生长状态和存活率进行生物统计分析,结合离体叶片的失水速率分析结果,显示OsWRKY95表现出正调控水稻抗旱性的功能。亚细胞定位结果显示,OsWRKY95定位于细胞核。酵母自激活实验结果显示,OsWRKY95存在自激活现象,表明OsWRKY95为转录激活子。4.为了探究OsWRKY95调控水稻抗旱的分子机制,以OsWRKY95的突变体植株和中花11号野生型植株为材料,进行转录组测序分析。结果显示,与野生型植株相比,其中91条基因在根部表达明显上调,51条表达下降。而在地上部组织中,有114个基因显著上调,25个基因显著下调。多个具有W-box基序的基因在突变体中显著下调,推测其可能为OsWRKY95潜在的靶基因,q PCR结果证实其在OsWRKY95的突变体中表达量降低。GO集分析显示,突变体中显著下调的基因在分子功能类别中的水解酶活性以及几丁质酶活性等功能上显著富集,KEGG通路富集分析显示,突变体中显著下调的基因在半乳糖代谢和MAPK通路上显著富集,推测OsWRKY95可能参与相关的信号通路,且可能与植物生物胁迫功能相关。5.酵母cDNA文库筛选互作蛋白试验结果显示,OsWRKY95转录因子与水稻磷饥饿应答4(Os PHR4)以及Os01g0974100蛋白互作。q PCR结果表明Os PHR4和Os01g0974100均受干旱胁迫诱导,表明OsWRKY95可能协同Os PHR4和Os01g0974100参与水稻的干旱调控。更多还原

【Abstract】 As one of the most important food crops,rice has a great demand for water during the whole growth period.However,water resources available for agriculture are generally scarce around the world.Under this background,the growth and development of rice and its high and stable yield have been severely challenged,and drought has become the main restricting factor.Therefore,it is of great significance for rice breeding to explore new genes and molecular mechanisms of drought resistance.Plant WRKY transcription factors play an important role in plant defense against abiotic and biological stresses.In this research,we investigated the function of OsWRKY95,a transcription factor in rice,in drought resistance of rice,and the main results were as follows:1.Bioinformatics analysis and haplotype analysis of OsWRKY95 showed that OsWRKY95 belonging to the WRKY transcription factor class Ⅲ family,the OsWRKY95 protein is composed of 260 amino acids and contains a conserved WRKY domain.The OsWRKY95 promoter sequence has a variety of cis-acting elements associated with abiotic stress.Haplotypes of OsWRKY95 were analyzed using 3K SNP data set of rice.The results showed that there were 6 haplotypes of OsWRKY95 and there was obvious differentiation between indica and japonica among different rice subspecies.Among them,Hap 1 was mainly distributed in japonica rice,and the other haplotypes were mainly distributed in indica rice.2.In order to explore whether OsWRKY95 is related to plant stress function,the expression of OsWRKY95 was induced by a variety of abiotic stresses and hormone treatments by fluorescence quantitative PCR.OsWRKY95 can not only respond to drought,cold and salt stress induction,but also be induced by ABA,JA,IAA and other hormones.Under the conditions of drought,cold,IAA and ABA treatments,the expression of OsWRKY95 first increased and then decreased.Under the conditions of salt stress and JA treatment,the expression level of OsWRKY95 continued to increase within 24 hours,and it was speculated that OsWRKY95 may participate in the regulation of rice growth and development,biotic stress and abiotic stress through ABA and JA signaling pathways.At the same time,the OsWRKY95 gene was constitutively expressed in rice,that is,it was expressed in both juvenile and adult tissues,and the expression level was the highest in panicle.3.The drought resistance function of OsWRKY95 overexpressed and knocked out plants was further identified at seedling stage.After simulated drought stress with 20%PEG6000 and restoration with Kimura B nutrient solution,the growth state and survival rate of seedlings were analyzed biostatistical,combined with the analysis results of water loss rate of isolated leaves.It was found that OsWRKY95 had a positive effect on drought resistance.The results of subcellular localization showed that OsWRKY95 was localized to the nucleus.The results of yeast self-activation experiments showed that OsWRKY95 had self-activation,indicating that OsWRKY95 was a transcriptional activator.4.To investigate the mechanism of OsWRKY95’s resistance to drought in rice,transcriptome was carried out in OsWRKY95 mutants and Zhonghua 11.The results indicated that there were significant up-regulation of 91 genes and 51 significant downregulation in the root of the mutant.In the aboveground tissues there were significant up-regulation of 114 genes and significant down-regulation of 25.and multiple genes with W-box motifs were significantly down-regulated in the mutants,suggesting that they may be potential target genes of OsWRKY95.q PCR results confirmed that its expression in OsWRKY95 mutants was reduced.GO analysis showed that the significantly down-regulated genes in the mutant were significantly enriched in the functions of hydrolase activity and chitinase activity in the molecular function category,and KEGG pathway enrichment analysis showed that the significantly downregulated genes in the mutant were significantly enriched in galactose metabolism and MAPK pathway,suggesting that OsWRKY95 may be involved in related signaling pathways,and may be related to the stress function of plant.5.Yeast screening cDNA library screening interaction protein assay results showed that OsWRKY95 transcription factor interacted with phosphate starvation response 4(Os PHR4)and Os01g0974100.The q PCR results showed that both Os PHR4 and Os01g0974100 were induced by drought stress,suggesting that OsWRKY95 may be involved in drought regulation in rice in conjunction with Os PHR4 and Os01g0974100.更多还原