【作者】 曲善民；

【导师】 郑殿峰；

【作者基本信息】 黑龙江八一农垦大学 ， 作物栽培学与耕作学， 2020， 博士

【摘要】 如何打破短期积水对大豆的胁迫伤害,是农业科研攻关的重要课题之一。本研究以大豆垦丰14和垦丰16为试验材料,R1期淹水胁迫,叶喷S3307为调控手段,采用盆栽控制试验,平行测定了两品种大豆生物学性状与产量构成、光合作用参数与物质积累、关键保护酶活性与内源激素含量、渗透调节物质含量与糖类含量、大豆籽粒氨基酸含量等生理生化指标,并观测了大豆根部与叶部的超显微结构,综合分析了大豆转录组系列数据,着重研究了S3307对大豆缓解淹水胁迫的调控效应和作用机理。主要结果如下:1.从生产性能看,R1期淹水胁迫降低大豆单株荚数、百粒重、干物质积累量,升高瘪荚率、瘪粒率,叶片容易早衰脱落,根量减少,造成大豆产量显著下降,减产幅度21.95%³9.58%。R1期淹水胁迫,叶面喷施S3307与正常供水CK相比,大豆垦丰14与垦丰16产量相近,S3307个别处理组生产性能甚至高于CK。叶面喷施50mg/LS3307可以显著遏制大豆瘪荚率、瘪粒率上升（p<0.01）,促进干物质累积,生产性能方面具有明显减损作用。2.从生长发育看,R1期淹水胁迫主要通过降低大豆根系侧根数量,减少根系伤流量,减少植株有效花果数,缩短功能叶片寿命,显著抑制了大豆的生长发育。R1期淹水胁迫,叶面喷施S3307与正常供水CK相比,大豆垦丰14与垦丰16株高均低于CK,但分枝较多,有效花果数较高。S3307调控组两个品种大豆叶片寿命均最长,光合时间最久,叶片持留性最好;根量最多,根系最发达;茎秆木质素、中性洗涤纤维含量最高;叶面喷施50mg/LS3307可以显著提高大豆灌浆能力,利于大豆健康生长,具有缓解淹水胁迫的作用。3.从生理指标看,R1期淹水胁迫,显著降低了大豆叶片气孔导度、叶片净光合速率、SPAD值、光合电子传递速率,造成大豆光合性能显著下降。淹水胁迫后,叶面喷施S3307两个大豆品种叶片净光合速率增加了15.36%⁴8.12%,光合电子传递速率随着光强增加上升最快,呈S型曲线递增。S3307调控组两个品种大豆SPAD值、胞间CO₂浓度、气孔导度、叶片蒸腾速率、净光合速率均有淹水+S3307组>CK组>淹水胁迫组的排序规律。叶面喷施50mg/LS3307对大豆减损具有生理保护作用。4.从生化指标看,R1期淹水胁迫,显著降低了大豆体内抗氧化酶活性、大豆叶片相关蛋白质丰度水平低下、三羧酸循环关键酶丰度水平较低、可溶性糖含量下降、激素水平失衡、糖类含量较低。S3307调控组两个品种大豆蔗糖、可溶性糖、果糖、淀粉含量均显著提升（p<0.05）,抗氧化酶SOD和POD活性显著升高（p<0.05）,MDA含量明显降低,可溶性蛋白和脯氨酸含量增加。叶面喷施50mg/LS3307有利于促进淹水胁迫下大豆三羧酸循环关键酶丰度增加,MDH、IDH、PDH丰度排序依次有W+S>CK>W;促进了GA3、IAA、ABA的应激响应。5.从显微结构看,淹水胁迫大豆根部细胞膜、各种细胞器完整性均不同程度受到破坏,细胞壁变薄,形成层细胞排列不规则,基粒紊乱,多数细胞核降解,线粒体撕裂破碎状增多,细胞器多以残体为主;S3307调控组大豆根部细胞膜完整性、各种细胞器完整性具有良好的保护效应,线粒体、质体,相对规则有序。淹水胁迫大豆叶部叶肉细胞、栅栏组织、细胞膜完整性均不同程度受到破坏,叶绿体类囊体肿胀破损,基粒紊乱,多数细胞核降解,一些线粒体嵴消失,叶绿体内、外膜消失,叶绿体撕裂破碎状增多,细胞内叶绿体、线粒体以残体为主;S3307调控组大豆叶部叶肉细胞、栅栏组织、细胞膜完整性、各种细胞器完整性具有良好的保护效应,叶绿体、线粒体、淀粉粒小而多,规则有序。6.从转录组分析看,垦丰14共有11974个基因受到激素和淹水胁迫影响,上调表达基因5454个,下调表达基因6519个。垦丰16共有9787个基因受到激素和淹水胁迫影响,上调表达基因5102个,下调表达基因4685个。喷施S3307之后,大豆有很多上调表达基因被富集在光合作用通路、脂代谢通路、抗氧化相关代谢途径;并发现丙酮酸激酶更多表达,减少了大豆厌氧代谢的乙醇积累,减轻自毒伤害;苯丙素类和黄酮类合成基因上调表达,抗坏血酸结合能力上调,增强细胞内细胞器修饰与功能运转能力;而在下调基因表达当中,碳代谢、氮代谢和脂质代谢都受到了严重影响,这与生产、生理、生化数据是吻合的。由qPCR分析,大豆不同处理组丙酮酸激酶基因的表达量:淹水+S3307组>正常水分CK>淹水胁迫,S3307对于减少丙酮酸生成乙醇路径具有调控作用;DNA损伤修复途径主要由核苷酸切除修复模式来主导完成;在FC≥2且FDR<0.01范围内高差异性目标表达基因中筛选功能描述与光合作用密切相关基因4个Glyma.15G221300/Glyma.02G104600[垦丰14],Glyma.02G156800/Glyma.01G198100[垦丰16],推测上述4个DEGs是造成提高净光合速率显著差异表达,产生抗逆性的原因所在。综上所述,施用S3307加强了对淹水大豆的定向调控水平,调节大豆良好生长发育,改善大豆生产性能,为大豆抗逆效应稳定表达起到了针对性作用,有效对冲淹水胁迫的危害性,最终实现大豆短期淹水胁迫下的抗逆机制的响应,施用S3307抗逆栽培前景广阔。更多还原

【Abstract】 In this study,two soybean varieties,Glycine max L.Kenfeng 14 and Glycine max L.Kenfeng 16,were subjected to watedogging at R1 growth stage,then leaf sprayed uniconazole(S3307)was used as control means withing pot control test.The following data of the two soybean varieties were determined in parallel: biological characteristics,yield components,photosynthetic parameters,material accumulation,key protective enzyme activity,endogenous hormone content,osmotic regulating material content,sugar content,amino acid content in soybean seeds and other physiological and biochemical indexes,meanwhile the soybean root and leaf vegetative organs ultra microstructure were determined,and soybean transcriptome data were also comprehensively analyzed to emphatically study the regulatory effect and the mechanism of S3307 on alleviating waterlogging stress of soybean.The main results were as follows:1.From the perspective of production performance,when the two soybean varieties were subjected to waterlogging stress at R1 stage,the number of per plant pods,hundred-grain weight,dry matter accumulation were reduced,the rate of wilted pods and pyrene grains were increased,the leaves were easy to fall off,the roots weight were reduced,which resulted in a significant reduction of soybean production by 21.95%~39.58%.During waterlogging stress at R1 stage,the yields of Kenfeng 14 and Kenfeng 16 were similar by the CK(treated with normal water supply)and leaf sprayed S3307 treatments,the individual treatment production performance by S3307 treatment was even higher than CK treatment.Leaves sprayed with 50 mg/L S3307 treatment significantly reduced the wilted pods rate(p<0.01),significantly increased the pyrene grains rate(p<0.01),promoted the dry matter accumulation and inhibited the soybean yield decrease.2.From the perspective of growing development,when the two soybean varieties were subjected to waterlogging stress at R1 stage,it mainly reduced the lateral roots number,root injury flow and the effective flowers and fruits number,shortened the functional leaves life,which finally significantly inhibited the soybean growing development.Compared with CK(treated with normal water supply)during waterlogging stress at R1,leaf sprayed SS3307 treatment decreased the height of Kenfeng 14 and Kenfeng 16,increased the number of branches and the effective number of flowers and fruits.Under S3307 treatment,the two soybean varieties with the longest leaf life,longest photosynthetic time and longest retention ability;with strong root system;with high content of stems lignin and neutral detergent fiber.Leaves sprayed with 50 mg/L S3307 treatment significantly improved the soybeans filling ability which was benefit to the soybeans healthy growth and relieve the damaging from waterlogging stress.3.From the perspective of physiological indicators,when the two soybean varieties were subjected to waterlogging stress at R1 stage,it significantly reduced the leaves stomatal conductance,leaf net photosynthetic rate,SPAD value,and photosynthetic electron transfer rate,resulting in a significantly decreased in soybean photosynthetic performance.Leaf sprayed S3307 treatment increased the leaves net photosynthetic rate by 15.36% ~ 48.12%;the photosynthetic electron transfer rate increased fastest with the increasing of light intensity and showed an S-shaped curve.The SPAD value,intercellular CO2 concentration,stomatal conductance,leaf transpiration rate,and net photosynthetic rate of the two soybean varieties changing rules in different treatments were as below: waterlogging stress+S3307group> CK group> waterlogging stress group.Leaf sprayed with 50mg/L S3307 played a physiological role in protecting soybeans from waterlogging damaging.4.From the perspective of biochemical indicators,when the two soybean varieties were subjected to waterlogging stress at R1 stage,it significantly reduced the antioxidant enzymes activity,reduced the abundance level of related protein in soybean leaves,reduced the abundance level of key enzymes in the tricarboxylic acid cycle,reduced the soluble sugar and sugar content,also resulted in hormone imbalance.The content of sucrose,soluble sugar,fructose,and starch in two varieties with S3307 teratment were significantly increased(p<0.05),the activities of antioxidant enzymes SOD and POD were significantly increased(p<0.05),the content of MDA was significantly reduced(p<0.05),the soluble protein and proline content were increased.Leaf sprayed with 50mg/L S3307 significantly improved the abundance of key enzymes in soybean tricarboxylic acid cycle,the MDH,IDH and PDH were as following: waterlogging stress+S3307group> CK group> waterlogging stress group;meanwhile,S3307 promoted the stress response of GA3,IAA and ABA.5.From the perspective of microstructure: when the two soybean varieties were subjected to waterlogging stress,the root cell membrane and various organelles integrity were damaged,the cell wall became thin,the formation layer cells were irregularly arranged,the basal granules were disordered,most of the nuclei were degraded,the number of mitochondrial tears and fragments were increased,and most organelles became residues.The S3307 control treatment had a good protective effect on the integrity of cell membrane and organelles of soybean root,the mitochondria and plastids were relatively regulated.When the two soybean varieties were subjected to waterlogging stress,the mesophyll cells,palisade tissue and cell membrane of leaves were damaged in varying degrees;the chloroplast thylakoid was swelled and damaged,the granule was disordered,most of the nucleus were degraded,some mitochondrial cristae were lost,the inside and outer membrane of the chloroplast were disappeared,the number of mitochondrial tears and fragments were increased,and most organelles became residues.the chloroplast was torn and broken,and the chloroplast and mitochondria were mainly residual.The S3307 control treatment had a good protective effect on the mesophyll cells,palisade tissues,membrane integrity and organelle integrity of soybean leaf.Chloroplasts,mitochondria and starch grains were small and orderly.6.From the perspective of transcriptome analysis,a total of 11974 genes involved in hormones and waterlogging stress were identified from Kenfeng 14,among these genes,5454up-and 6519 down-regulated genes were presented.A total of 9787 involved in hormones and waterlogging stress were identified from Kenfeng 16,among these genes,5102 up-and 4685down-regulated genes were presented.After spraying S3307,more up-regulated genes were enriched in photosynthesis pathway,lipid metabolism pathway,and antioxidant-related metabolic pathway.Besides,more pyruvate kinase was expressed,the accumulation of ethanol in anaerobic metabolism was reduced,the root autotoxicity was reduced;Synthetic genes of phenylpropanoids and flavonoids were up-regulated,ascorbic acid binding capacity was up-regulated,cell organelles modify and function ability were enhanced.Among down-regulated expressed genes,carbon metabolism,nitrogen metabolism and lipid metabolism had been severely affected,which were consistent with the production,physiological,and biochemical data.According to q PCR analysis,the pyruvate kinase genes presented in different treatments of soybean were as following order: waterlogging stress+S3307treatment> CK treatment >waterlogging stress treatment,S3307 had a regulatory effect on reducing the path of pyruvate to ethanol;The DNA damage repair pathway was mainly dominated by nucleotide excision repair pattern;4 closely related to photosynthesis genes were selected in the range of highly differential target expressed genes with FC ≥ 2 and FDR<0.01,they were 15G221300/Glyma.02G104600[Kenfeng 14],Glyma.02G156800/Glyma.01G198100 [Kenfeng16],respectively.It is speculated that the above four DEGs were responsible for the significant differential expression of improving net photosynthetic rate and stress resistance.In summary,the application of S3307 strengthens the directional regulation,promotes the soybean growth and development,improves the soybean production performance,plays a targeted role in the stable expression of soybean anti-stress effect,effectively hedge against the hazard of waterlogging stress,which finally illuminate the resistance mechanism of soybean under short-term waterlogging stress.The application of S3307 has a broad prospect in anti-stress cultivation.更多还原