【作者】 王倩；

【导师】 魏赛金； 彭帅英； 戴斌胜；

【作者基本信息】 江西农业大学 ， 生物与医药（专业学位）， 2025， 硕士

【摘要】 生物活性肽在植物抗逆中具有重要作用,盐碱地的广泛分布和豇豆（Vigna unguiculata）的重要经济价值使得生物活性肽促进豇豆的耐盐胁迫研究具有重要的理论和实际意义。发酵豆制品因其富含大豆蛋白和微生物代谢产物,成为重要的活性肽来源,因此,本研究以发酵豆制品（腐乳、豆豉、臭豆腐、纳豆和豆瓣酱）为原料,从中筛选高效产生物活性肽的菌株,通过全基因组测序及生信分析技术初步探究其产生物活性肽的分子基础,通过单因素试验和响应面法优化确定其最佳的产肽条件,并进一步研究生物活性肽缓解盐胁迫对豇豆幼苗的抑制效果:1.以发酵豆制品（腐乳、豆豉、臭豆腐、纳豆和豆瓣酱）为原料,先后通过酪蛋白水解圈初筛及ACE酶抑制活性复筛,获得一株高效产生物活性肽的菌株CDF17。通过菌落形态、生理生化鉴定和分子生物学分析,鉴定该菌株为枯草芽孢杆菌（Bacillus subtilis）,命名为B.subtilis CDF17,并测定了菌株的生长特性。2.通过全基因组测序技术获得B.subtilis CDF17的全基因组信息:基因组大小为4,254,884 bp,GC含量为44%,包含4,312个蛋白编码基因,编码基因长度占基因组总长度的88%。基因组功能注释结果表明,B.subtilis CDF17的基因主要参与碳水化合物转运与代谢、翻译、核糖体结构与生物合成等生物学过程,且在新陈代谢相关通路中表现出较高的活性。此外,基因组中未检测到毒力因子,表明该菌株具有较高的安全性。通过对菌株全基因组信息进行蛋白酶与肽酶基因生信分析,共挖掘到20个基因编码蛋白酶和肽酶编码基因,包括细胞外蛋白酶、ATP依赖的蛋白酶、Lon蛋白酶、膜蛋白酶、信号肽酶和肽酶,说明B.subtilis CDF17具备水解蛋白质底物的分子基础。3.通过单因素试验和响应面法优化发酵条件,确定B.subtilis CDF17最适产肽条件如下:发酵时间12.5 h、盐度3.0%、初始pH值7.9、发酵温度36℃、接种量5%。在此条件下,发酵液中短肽含量达到16.00 mg/mL,较优化前的9.79 mg/mL显著提高了63.43%。这一结果表明,通过对发酵条件进行全面优化,能显著提升B.subtilis CDF17的产生物活性肽能力。4.通过在300 mM盐胁迫环境下,研究B.subtilis CDF17所产生物活性肽对两种豇豆品种（丰产2号与夏宝2号）幼苗耐受盐胁迫的影响,并对其生理生化机制进行了初步的探究,结果表明B.subtilis CDF17所产生物活性肽能有效缓解盐胁迫对两种豇豆的抑制作用并通过以下方式提高豇豆对盐胁迫的耐受能力:（1）显著提高豇豆的净光合速率,尤其是丰产2号的净光合速率提高了170.62%;（2）显著降低豇豆叶片中ROS含量包括超氧阴离子（O₂^-·）和过氧化氢（H₂O₂）含量,以及提高抗氧化酶（SOD、POD、CAT）活性,从而缓解了豇豆叶片的氧化损伤;（3）提高豇豆叶片中的游离脯氨酸和可溶性蛋白含量,帮助豇豆维持细胞内的渗透平衡,增强其抗逆性;（4）降低豇豆茎和叶中Na⁺的积累以及促进K⁺、Ca²⁺和Mg²⁺的吸收,使细胞内的Na⁺/K⁺比值维持在较低水平,从而增强了豇豆对盐胁迫的耐受性。综上所述,本研究从发酵豆制品中分离到一株高产生物活性肽的菌株,经鉴定后确定其为B.subtilis菌株;通过对其产肽条件进行响应面优化,使其产生物活性肽含量显著提高;此外,B.subtilis CDF17所产生物活性肽显著提高了盐胁迫下豇豆幼苗的净光合速率,有机渗透物质的含量以及抗氧化酶活性,缓解了盐胁迫引起的离子稳态失衡,从而增强豇豆幼苗对盐胁迫的耐受性。B.subtilis CDF17及其所产的生物活性肽在农业和食品工业中具有广泛的应用潜力。更多还原

【Abstract】 Bioactive peptides play a crucial role in plant stress resistance.The widespread distribution of saline-alkali soils and the significant economic value of cowpea（Vigna unguiculata）make the study of bioactive peptides in enhancing cowpea’s salt stress tolerance highly important,both theoretically and practically.Fermented soybean products,rich in soybean proteins and microbial metabolites,serve as a major source of bioactive peptides.Therefore,this study used fermented soybean products（fermented tofu,douchi,stinky tofu,natto,and broad bean paste）as raw materials to screen for strains with high bioactive peptide production.Whole-genome sequencing and bioinformatics analysis were employed to preliminarily explore the molecular basis of bioactive peptide production.Single-factor experiments and response surface methodology were used to optimize peptide production conditions,followed by further investigation into the effects of bioactive peptides in alleviating salt stress-induced inhibition in cowpea seedlings:1.Using fermented soybean products（fermented tofu,douchi,stinky tofu,natto,and broad bean paste）as raw materials,a strain CDF17 with high bioactive peptide production was isolated through initial screening via casein hydrolysis zones and secondary screening based on ACE enzyme inhibitory activity.Through colony morphology,physiological and biochemical identification,and molecular biological analysis,the strain was identified as Bacillus subtilis and named B.subtilis CDF17.The growth characteristics of the strain were also determined.2.Whole-genome sequencing revealed the genomic information of B.subtilis CDF17:a genome size of 4,254,884 bp,GC content of 44%,and 4,312 protein-coding genes,accounting for 88%of the total genome length.Functional annotation indicated that the genes of B.subtilis CDF17 are primarily involved in carbohydrate transport and metabolism,translation,ribosomal structure and biogenesis,and other biological processes,with high activity in metabolic pathways.Additionally,no virulence factors were detected,suggesting the strain is likely non-pathogenic.Bioinformatics analysis of protease and peptidase genes identified 20 genes encoding extracellular proteases,ATP-dependent proteases,Lon proteases,membrane proteases,signal peptidases,and peptidases,confirming the molecular basis for B.subtilis CDF17’s ability to hydrolyze protein substrates.3.Single-factor experiments and response surface methodology were used to optimize fermentation conditions.The optimal peptide production conditions for B.subtilis CDF17were determined as follows:fermentation time of 12.5 h,salinity of 3.0%,initial p H of 7.9,fermentation temperature of 36°C,and inoculation volume of 5%.Under these conditions,the short peptide content in the fermentation broth reached 16.00 mg/m L,a significant increase of 63.43%compared to the pre-optimization level of 9.79 mg/m L.These results demonstrate that comprehensive optimization of fermentation conditions can significantly enhance the bioactive peptide production capacity of B.subtilis CDF17.4.Under 300 mM salt stress,the effects of bioactive peptides produced by B.subtilis CDF17 on the salt stress tolerance of two cowpea varieties（Fengchan No.2 and Xiabao No.2）were studied,and the physiological and biochemical mechanisms were preliminarily explored.The results showed that the bioactive peptides effectively alleviated salt stress-induced inhibition in both cowpea varieties and enhanced their salt stress tolerance through the following mechanisms:（1）Significantly increasing the net photosynthetic rate of cowpea,particularly in Fengchan No.2,where the rate increased by 170.62%;（2）Markedly reducing reactive oxygen species（ROS）content,including superoxide anion（O₂^-·）and hydrogen peroxide（H₂O₂）,while enhancing the activity of antioxidant enzymes（SOD,POD,CAT）,thereby mitigating oxidative damage in cowpea leaves;（3）Increasing the content of free proline and soluble proteins in cowpea leaves,helping to maintain intracellular osmotic balance and improve stress resistance;（4）Reducing Na⁺accumulation in stems and leaves while promoting the absorption of K⁺,Ca²⁺,and Mg²⁺,thereby maintaining a low intracellular Na⁺/K⁺ratio and enhancing salt stress tolerance.In summary,this study isolated a high bioactive peptide-producing strain from fermented soybean products,identified as B.subtilis.Response surface optimization of peptide production conditions significantly increased the bioactive peptide yield.Furthermore,the bioactive peptides produced by B.subtilis CDF17 significantly improved the net photosynthetic rate,organic osmolyte content,and antioxidant enzyme activity of cowpea seedlings under salt stress,alleviating ion homeostasis imbalance caused by salt stress and thereby enhancing salt stress tolerance.B.subtilis CDF17 and its bioactive peptides exhibit broad application potential in agriculture and the food industry.更多还原