【作者】 陈静；

【导师】 黄琳凯； 王赞；

【作者基本信息】 四川农业大学 ， 草学， 2020， 硕士

【摘要】 多倍化是植物进化过程中主要的动力之一,植物多倍化后在表型、生态适应性和抗病抗逆性等方面发生变化,对农业生产及研究具有重要作用。紫花苜蓿作为世界范围内栽培最广的豆科牧草,拥有较高的营养价值和经济效益,而干旱成为影响其产量、限制其大面积栽种的重要因素之一,故了解紫花苜蓿抗旱机制,对遗传改良、新品种培育变得尤为重要。本研究以59份栽培紫花苜蓿(Medicago sativa ssp.sativa,2n=4X=32)与18份祖先种蓝花苜蓿(Medicago sativa ssp.caerulea,2n=2X=16)为材料,在对77份种质材料进行抗旱性评价鉴定的基础上,选择ZW0443(紫花苜蓿)及ZW0023(蓝花苜蓿)两份材料在干旱胁迫第6、12、16 d进行生理及转录组测序研究,以期揭示二者在生理和转录组水平上响应干旱胁迫的异同。主要结果如下:1.利用聚类和主成分分析对77份种质材料进行抗旱性研究表明,供试材料被划分为抗旱性存在差异的两个独立类群,即紫花苜蓿和蓝花苜蓿,而且紫花苜蓿群体的抗旱性强于蓝花苜蓿群体。2.通过研究紫花苜蓿(ZW0443)和蓝花苜蓿(ZW0023)植株对干旱胁迫的生理响应,发现在干旱胁迫下,两份材料叶片相对含水量均呈下降趋势,且ZW0023下降的幅度小于ZW0443,说明ZW0023叶片具有相对较强的保水能力;ZW0023叶片丙二醛含量显著高于ZW0443,表明ZW0443膜系统受损伤的程度低于ZW0023;ZW0443和ZW0023在干旱胁迫下体内都积累了脯氨酸及脱落酸,且ZW0443脯氨酸含量增加的幅度大于ZW0023。3.通过转录组测序结果发现,ZW0443在干旱胁迫第6、12、16 d分别鉴定了746、3530、4226个差异表达基因(DEGs),ZW0023分别鉴定了720、2184、2142个DEGs;ZW0443和ZW0023相比,在正常浇水第6、12、16 d分别发现1566、1372、3647个DEGs,在干旱处理第6、12、16 d分别发现1627、1739、4390个DEGs。GO分析表明以上DEGs主要参与了细胞过程、代谢过程、单生物过程、对刺激的反应等生物学过程。KEGG富集分析表明,植物激素信号转导途径在ZW0443及ZW0023中均显著富集,但ZW0443显著富集的胁迫时间点要早于ZW0023;淀粉和蔗糖代谢、维生素B6代谢途径仅在ZW0023中显著富集,而MAPK信号通路-植物、光合作用-天线蛋白、光合作用等途径仅在ZW0443中显著富集。另外,我们发现在干旱胁迫第12 d的渗透物质代谢途径中,ZW0023可能主要通过调控与淀粉分解、海藻糖及葡萄糖合成途径相关基因的表达以应对干旱胁迫,而ZW0443可能主要通过调控与蔗糖、果糖、海藻糖及棉子糖代谢途径相关基因的表达以应对干旱胁迫。更多还原

【Abstract】 Polyploidy is one of the main driving forces in the process of plant evolution.Plants have changes in phenotypic,ecological adaptability,disease resistance and stress resistance after being polyploidy,which plays an important role in agricultural production and research.Alfalfa with high nutritional value and economic benefits is the most widely cultivated legume forage in the world.However,drought is one of the important limited factors affecting its yield and large-scale cultivation.Therefore,it is considerably important for genetic improvement and new variety cultivation to understand the mechanism of drought resistance in alfalfa.The research evaluated the drought resistance of 59 autotetraploid alfalfa(M.sativa ssp.sativa)and 18 diploid ancestor(M.sativa ssp.caerulea)materials,ZW0443(ssp.sativa)and ZW0023(ssp.caerulea)were selected for next physiological and transcriptome sequencing studies on the 6th,12th and 16th days of drought stress,in order to reveal the similarities and differences of two materials responsing to drought stress at the physiological and transcriptome levels.The main results are as follows:1.The drought resistance of 77 germplasm materials were studied by cluster and principal component analysis,and it was found that these materials were divided into two independent groups with different drought resistance,ssp.sativa and ssp.caerulea.Aditionally,autotetraploid ssp.sativa performs better drought resistance than diploid ancestor ssp.caerulea.2.Physiological changes of two materials under drought stress were studied.The relative water content of the leaves of ZW0443 and ZW0023 significantly reduced under drought stress,and the decrease of ZW0023 was slighter than ZW0443,which indicates that the leaves of ZW0023 have relatively strong water retention capacity.The malondialdehyde content of ZW0023 was significantly higher than ZW0443 under drought stress,indicating that the damage of membrane system in ZW0443 was less severe than ZW0023.Both of ZW0443 and ZW0023 accumulated proline and abscisic acid under drought stress,and the increase of proline in ZW0443 was higher than that in ZW0023.3.The results of transcriptome sequencing showed that there were 746,3530 and4226 differentially expressed genes(DEGs)in ZW0443 on the 6th,12th and 16th days of drought stress,and 720,2184 and 2142 DEGs in ZW0023,respectively.Compared ZW0443 with ZW0023,1566,1372 and 3647 DEGs were found on the 6th,12th and 16th days under normal watering condition,and 1627,1739 and 4390 DEGs were found on the6th,12th and 16th days under drought stress condition,respectively.GO enrichment analysis shows that these DEGs are mainly involved in cellular processes,metabolic processes,single-organism process,responses to stimuli and other biological processes.KEGG enrichment analysis showed that the plant hormone signal transduction pathways was significantly enriched in ZW0443 and ZW0023,but ZW0443 was significantly enriched earlier than ZW0023.The study found that starch and sucrose metabolism,vitamin B6 metabolism pathway were only significantly enriched in ZW0023,and MAPK signal pathway-plant,photosynthesis-antenna protein,photosynthesis and other pathways were only significantly enriched in ZW0443.On the 12th day under drought stress,we found that ZW0023 might respond to drought stress by regulating the expression of genes related to starch decomposition,trehalose and glucose synthesis pathway,while ZW0443might respond to stress by regulating the expression of genes involved in sucrose,fructose,trehalose and raffinose metabolism pathway.更多还原