Article(id=1309574993005076817, tenantId=1146029695717560320, journalId=1308397196074385509, issueId=1309574965775659485, articleNumber=null, orderNo=null, doi=10.11733/j.issn.1007-0435.2026.08.007, pmid=null, cstr=null, oa=null, hot=0, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=null, receivedDate=1759075200000, receivedDateStr=2025-09-29, revisedDate=1763654400000, revisedDateStr=2025-11-21, acceptedDate=null, acceptedDateStr=null, onlineDate=1790156609058, onlineDateStr=2026-09-23, pubDate=null, pubDateStr=null, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1790156609058, onlineIssueDateStr=2026-09-23, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1790156609058, creator=13701087609, updateTime=1790156609058, updator=13701087609, issue=Issue{id=1309574965775659485, tenantId=1146029695717560320, journalId=1308397196074385509, year='2026', volume='34', issue='8', pageStart='2753', pageEnd='3140', issueExtLink='null', onlineDate='null', pubDate='1786723200000', pubDateStr='2026-08-15', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=-1, specialIssue=null, createTime=1790156602567, creator='13701087609', updateTime=1790156935415, updator='13701087609', preIssue=null, nextIssue=null, articleTotal=null, ext={EN=IssueExt(id=1309576361992352087, tenantId=1146029695717560320, journalId=1308397196074385509, issueId=1309574965775659485, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1309576361992352088, tenantId=1146029695717560320, journalId=1308397196074385509, issueId=1309574965775659485, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null, downloadFileDto=null}, startPage=2826, endPage=2836, ext={EN=ArticleExt(id=1309574994565357909, articleId=1309574993005076817, tenantId=1146029695717560320, journalId=1308397196074385509, language=EN, title=Optimization of Pretreatment Methods for Metabolites of Rhizobia Isolated from Lespedeza davurica Using Box-Behnken Response Surface Methodology, columnId=null, journalTitle=Acta Agrestia Sinica, columnName=null, runingTitle=null, highlight=null, articleAbstract=The symbiont formed by legumes and rhizobia plays a crucial role in the nitrogen cycle, and root exudates significantly affect the nodulation process. The pretreatment and detection methods for metabolites of rhizobia from Lespedeza daurica have not been systematically optimized. In this study, the extraction efficiency of rhizobial metabolites was improved by optimizing pretreatment conditions, including ultrasonic temperature (A), ultrasonic time (B), derivatization temperature (C), and derivatization time (D). Single-factor experiments were used to determine the ranges of detection conditions, followed by a four-factor, three-level Box-Behnken response surface methodology (ultrasonic temperature: 30-40℃ ultrasonic time: 7-11 min derivatization time: 20-100 min derivatization temperature: 50-90℃) for optimization design. A quadratic regression model was established to determine the optimal conditions and verify their reliability. The optimal conditions determined by single-factor experiments were ultrasonic temperature 35℃, ultrasonic time 9 min, derivatization temperature 70℃, and derivatization time 60 min. The response surface model (R2=0.9035) showed that the linear terms A and C, the interaction term CD, and the quadratic terms A2, C2, and D2 had extremely significant effects on the results (P<0.01), while the interaction term AD had a significant effect (P<0.05). After optimization, 145 metabolites were detected, representing an increase of 198.60% compared with the pre-optimization level, with an error of 3.5%. Methodological validation showed that the peak area RSD of the internal standard L-2-chloro-phenylalanine was 8.2% (n=6), and the signal-to-noise ratio (S∶N) was 9.9∶1, which met the requirements for metabolomic detection. This study established a pretreatment method for the detection of metabolites from rhizobia of Lespedeza daurica, providing a technical basis for their extraction and analysis., authors=LI Yi-juan1,2,3, BAI Yu-fang1,2,3, CHEN Yu-chong1,2,3, MA Jie4, YANG Kai-yuan1,2,3, CUI Qing-liang4, ZHAO Xiang1,2,3, authorsList=LI Yi-juan, BAI Yu-fang, CHEN Yu-chong, MA Jie, YANG Kai-yuan, CUI Qing-liang, ZHAO Xiang, authorCompany=1 College of Grassland Science, Shanxi Agricultural University, Taigu, Shanxi Province 030800, China; 2 Shanxi Key Laboratory of Grassland Ecological Protection and Native Grass Germplasm Innovation, Taigu, Shanxi Province 030801, China; 3 Youyu Loess Plateau Grassland Ecosystem National Research Station, Youyu, Shanxi Province 037200, China; 4 College of Agricultural Engineering, Shanxi Agricultural University, Taigu, Shanxi Province 030800, China, correspAuthors=null, authorNote=null, correspAuthorsNote=null, copyrightStatement=null, copyrightOwner=null, extLink=null, articleAbsUrl=null, sourceXml=null, magXml=null, pdfUrl=null, pdf=null, pdfFileSize=null, pdfExtLink=null, richHtmlUrl=null, mobilePdfUrl=null, reviewReport=null, pdfFirstPage=null, abstractGraph=null, abstractGraphContent=null, abstractVideo=null, citation=null, cebUrl=null, magXmlContent=null, mapNumber=null, fund=null), CN=ArticleExt(id=1309574994452111699, articleId=1309574993005076817, tenantId=1146029695717560320, journalId=1308397196074385509, language=CN, title=Box-Behnken响应面法优化达乌里胡枝子根瘤菌代谢产物的前处理方法, columnId=1309565024058835950, journalTitle=草地学报, columnName=研究论文, runingTitle=null, highlight=null, articleAbstract=豆科植物-根瘤菌共生体在氮循环中至关重要,其根系代谢产物显著影响结瘤过程。达乌里胡枝子(Lespedeza daurica)根瘤菌代谢产物前处理检测方法尚未进行系统优化。通过优化前处理(超声温度A及时间B、衍生温度C及时间D),提高其代谢产物提取效率。采用单因素试验筛选检测条件范围,再采用四因素三水平Box-Behnken响应面法优化设计(超声温度30~40℃、超声时间7~11 min、衍生时间20~100 min、衍生温度50~90℃),建立二次回归模型确定最优条件并验证其可靠性。单因素试验确定检测条件:超声温度35℃、超声时间9 min、衍生温度70℃、衍生时间60 min;响应面模型(R²=0.9035)显示一次项A、C,交互项CD和二次项A²、C²、D²对结果影响极显著(P<0.01),交互项AD影响显著(P<0.05)。优化后共检测到代谢产物145个,较优化前提高了198.60%,误差3.5%;方法学验证:内标物质L-2-氯-苯丙氨酸的峰面积RSD为8.2%(n=6),信噪比(S∶N)=9.9∶1,符合代谢组学检测要求。确定达乌里胡枝子根瘤菌代谢产物检测前处理方法,为其提取与检测提供技术依据。, authors=李宜娟1,2,3, 白玉芳1,2,3, 陈禹翀1,2,3, 马洁4, 杨凯元1,2,3, 崔清亮4, 赵祥1,2,3, authorsList=李宜娟, 白玉芳, 陈禹翀, 马洁, 杨凯元, 崔清亮, 赵祥, authorCompany=1 山西农业大学草业学院, 山西 太谷 030800; 2 草地生态保护与乡土草种质创新山西省重点实验室, 山西 太谷 030801; 3 山西右玉黄土高原草地生态系统国家定位观测研究站, 山西 右玉 037200; 4 山西农业大学农业工程学院, 山西 太谷 030800, correspAuthors=null, authorNote=李宜娟(2000-),女,汉族,甘肃白银人,硕士研究生主要从事草地微生物学研究,E-mail:1328636321@qq.com, correspAuthorsNote=null, copyrightStatement=null, copyrightOwner=null, extLink=null, articleAbsUrl=null, sourceXml=null, magXml=null, pdfUrl=null, pdf=HnIbbxw4ulk+JjoJI94psw==, pdfFileSize=4387177, pdfExtLink=null, richHtmlUrl=null, mobilePdfUrl=null, reviewReport=null, pdfFirstPage=null, abstractGraph=null, abstractGraphContent=null, abstractVideo=null, citation=null, cebUrl=null, magXmlContent=null, mapNumber=null, fund=山西省重点研发计划项目 (2402140601011); 中央引导地方科技发展资金项目 (YDZJSX2024B008))}, authors=[Author(id=1320572912004878898, tenantId=1146029695717560320, journalId=null, articleId=1309574993005076817, orderNo=null, firstName=null, middleName=null, lastName=null, nameCn=null, orcid=null, stid=null, country=null, authorPic=null, dead=null, email=null, emailSecond=null, emailThird=null, correspondingAuthor=null, authorType=null, ext={EN=AuthorExt(id=null, tenantId=null, journalId=1308397196074385509, articleId=1309574993005076817, authorId=1320572912004878898, language=EN, stringName=LI Yi-juan, BAI Yu-fang, CHEN Yu-chong, MA Jie, YANG Kai-yuan, CUI Qing-liang, ZHAO Xiang, firstName=null, middleName=null, lastName=null, prefix=null, suffix=null, authorComment=null, nameInitials=null, affiliation=null, department=null, xref=null, address=null, bio=null, bioImg=null, bioContent=null, aboutCorrespAuthor=null), CN=AuthorExt(id=null, tenantId=null, journalId=1308397196074385509, articleId=1309574993005076817, authorId=1320572912004878898, language=CN, stringName=李宜娟, 白玉芳, 陈禹翀, 马洁, 杨凯元, 崔清亮, 赵祥, firstName=null, middleName=null, lastName=null, prefix=null, suffix=null, authorComment=null, nameInitials=null, affiliation=null, department=null, xref=null, address=null, bio=null, bioImg=null, bioContent=null, aboutCorrespAuthor=null)}, companyList=null)], keywords=[Keyword(id=1309574994754101590, tenantId=1146029695717560320, journalId=1308397196074385509, articleId=1309574993005076817, language=CN, orderNo=1, keyword=达乌里胡枝子), Keyword(id=1309574994842181975, tenantId=1146029695717560320, journalId=1308397196074385509, articleId=1309574993005076817, language=CN, orderNo=2, keyword=根瘤菌代谢产物), Keyword(id=1309574994921873754, tenantId=1146029695717560320, journalId=1308397196074385509, articleId=1309574993005076817, language=CN, orderNo=3, keyword=前处理方法优化), Keyword(id=1309574995060285787, tenantId=1146029695717560320, journalId=1308397196074385509, articleId=1309574993005076817, language=EN, orderNo=1, keyword=Lespedeza daurica), Keyword(id=1309574995144171868, tenantId=1146029695717560320, journalId=1308397196074385509, articleId=1309574993005076817, language=EN, orderNo=2, keyword=Metabolites of Rhizobia), Keyword(id=1309574995219669341, tenantId=1146029695717560320, journalId=1308397196074385509, articleId=1309574993005076817, language=EN, orderNo=3, keyword=Optimization of pretreatment methods)], refs=null, funds=null, companyList=null, figs=null, attaches=null, journal=Journal(id=1308395841968504927, delFlag=0, nameCn=草地学报, nameEn=Acta Agrestia Sinica, nameHistory1=null, nameHistory2=null, issn=1007-0435, eissn=null, cn=11-3362/S, coden=null, periodic=0, language=CN, oaType=null, ccby=null, superviseOffice=null, ownerOffice=null, pubOffice=null, editorOffice=null, officeType=null, aims=null, clcCode=null, officeProv=null, officeCity=null, officeAddr=null, officeZip=null, officeEmail=null, officePhone=null, editDirector=null, officeDirector=null, officeDirectorPhone=null, officeStaffNum=null, officeEmpNum=null, coverPicUrl=gCdi9sXCX82eLR6S1d4VLg==, journalPrice=null, startedYear=null, abbrevIsoEn=Acta Agrestia Sinica, journalRemark=null, publicationField=null, createdTime=1789875477545, updatedTime=1789876241473, createdBy=18614031015, updatedBy=13701087609, firstLetterCn=C, firstLetterEn=C, subjectCode=Agriculture, subjectName=null, subjectCodeEn=Agriculture, subjectNameEn=null, picCn=gCdi9sXCX82eLR6S1d4VLg==, picEn=4dKqEDfsaGuyf+bpm/3gfw==, jcr=null, cjcr=null, exts=[JournalExt(id=1308399046192812446, language=CN, name=草地学报, nameHistory1=null, nameHistory2=null, managedBy=, sponsoredBy=, publishedBy=, editorOffice=, officeProv=null, officeCity=null, officeAddr=, officeZip=, editDirector=, officeDirector=null, officePhone=null, coverPicUrl=null, journalRemark=, submitArticleUrl=null, websiteUrl=, createdTime=1789876241491, updatedTime=1789876241491, createdBy=13701087609, updatedBy=13701087609, submissionGuidelinesUrl=, submissionAuthorUrl=https://manu40.magtech.com.cn/JournalX_cdxb/authorLogOn.action, submissionEditorUrl=https://manu40.magtech.com.cn/JournalX_cdxb/editorLogOn.action, submissionReviewUrl=https://manu40.magtech.com.cn/JournalX_cdxb/expertLogOn.action, submissionCeEditorUrl=, submissionAeEditorUrl=, option={"copyright":""}), JournalExt(id=1308399046268309919, language=EN, name=Acta Agrestia Sinica, nameHistory1=null, nameHistory2=null, managedBy=, sponsoredBy=, publishedBy=, editorOffice=, officeProv=null, officeCity=null, officeAddr=, officeZip=, editDirector=, officeDirector=null, officePhone=null, coverPicUrl=null, journalRemark=, submitArticleUrl=null, websiteUrl=, createdTime=1789876241509, updatedTime=1789876241509, createdBy=13701087609, updatedBy=13701087609, submissionGuidelinesUrl=, submissionAuthorUrl=https://manu40.magtech.com.cn/JournalX_cdxb/authorLogOn.action, submissionEditorUrl=https://manu40.magtech.com.cn/JournalX_cdxb/editorLogOn.action, submissionReviewUrl=https://manu40.magtech.com.cn/JournalX_cdxb/expertLogOn.action, submissionCeEditorUrl=, submissionAeEditorUrl=, option={"copyright":""})], databaseList=null, tenantJournalId=1308397196074385509, websiteList=[Website(id=1308429426430071695, webName=null, webTitle=null, webDomain=null, webCopyrigh=null, webIpcNo=null, seoTitle=null, seoKeywords=null, seoDescription=null, tenantJournalId=null, journalId=1308397196074385509, journalNameCn=null, journalNameEn=null, grayFlag=null, tenantId=1146029695717560320, platformId=null, journalGroupId=null, journalGroupNameCn=null, journalGroupNameEn=null, type=1, domain=https://castjournals.cast.org.cn/joweb/cdxb/CN, language=CN, createTime=1789883484704, createBy=18614031015, updateTime=1789883745058, updateBy=18614031015, name=草地学报-中文, tplId=1146099689490845704, title=草地学报, delFlag=0, indexPage=/home, props=[WebsiteProps(id=1308430669353340972, tenantId=1146029695717560320, journalId=null, journalGroupId=null, siteId=1308429426430071695, code=articleTextType, value=kx, createTime=1789883781040, updateTime=1789883781040, creator=18614031015, updator=18614031015), WebsiteProps(id=1308430669328175145, tenantId=1146029695717560320, journalId=null, journalGroupId=null, siteId=1308429426430071695, code=banner, value=null, createTime=1789883781034, updateTime=1789883781034, creator=18614031015, updator=18614031015), WebsiteProps(id=1308430669382701103, tenantId=1146029695717560320, journalId=null, journalGroupId=null, siteId=1308429426430071695, code=grayFlag, value=0, createTime=1789883781047, updateTime=1789883781047, creator=18614031015, updator=18614031015), WebsiteProps(id=1308430669319786536, tenantId=1146029695717560320, journalId=null, journalGroupId=null, siteId=1308429426430071695, code=logo, value=https://castjournals.cast.org.cn/joweb/cdxb/CN/file/pic?fileId=BZ28lIoKdB54MmA0z6K66g==, createTime=1789883781032, updateTime=1789883781032, creator=18614031015, updator=18614031015), WebsiteProps(id=1308430669395284017, tenantId=1146029695717560320, journalId=null, journalGroupId=null, siteId=1308429426430071695, code=minRunFlag, value=0, createTime=1789883781050, updateTime=1789883781050, creator=18614031015, updator=18614031015), WebsiteProps(id=1308430669344952363, tenantId=1146029695717560320, journalId=null, journalGroupId=null, siteId=1308429426430071695, code=picServerUrl, value=https://castjournals.cast.org.cn/joweb/cdxb/CN/file/pic, createTime=1789883781038, updateTime=1789883781038, creator=18614031015, updator=18614031015), WebsiteProps(id=1308430669391089712, tenantId=1146029695717560320, journalId=null, journalGroupId=null, siteId=1308429426430071695, code=silenceFlag, value=0, createTime=1789883781049, updateTime=1789883781049, creator=18614031015, updator=18614031015), WebsiteProps(id=1308430669336563754, tenantId=1146029695717560320, journalId=null, journalGroupId=null, siteId=1308429426430071695, code=staticResourcePath, value=https://castjournals.cast.org.cn/joweb/cast_kjdb_cn_619/, createTime=1789883781036, updateTime=1789883781036, creator=18614031015, updator=18614031015), WebsiteProps(id=1308430669361729581, tenantId=1146029695717560320, journalId=null, journalGroupId=null, siteId=1308429426430071695, code=themeColor, value=null, createTime=1789883781042, updateTime=1789883781042, creator=18614031015, updator=18614031015), WebsiteProps(id=1308430669370118190, tenantId=1146029695717560320, journalId=null, journalGroupId=null, siteId=1308429426430071695, code=themeStyle, value=null, createTime=1789883781044, updateTime=1789883781044, creator=18614031015, updator=18614031015)]), Website(id=1308429426501374870, webName=null, webTitle=null, webDomain=null, webCopyrigh=null, webIpcNo=null, seoTitle=null, seoKeywords=null, seoDescription=null, tenantJournalId=null, journalId=1308397196074385509, journalNameCn=null, journalNameEn=null, grayFlag=null, tenantId=1146029695717560320, platformId=null, journalGroupId=null, journalGroupNameCn=null, journalGroupNameEn=null, type=1, domain=https://castjournals.cast.org.cn/joweb/cdxb/EN, language=EN, createTime=1789883484721, createBy=18614031015, updateTime=1789883716139, updateBy=18614031015, name=草地学报-英文, tplId=1146101810881728533, title=Acta Agrestia Sinica, delFlag=0, indexPage=/home, props=[WebsiteProps(id=1308430635979264030, tenantId=1146029695717560320, journalId=null, journalGroupId=null, siteId=1308429426501374870, code=articleTextType, value=kx, createTime=1789883773083, updateTime=1789883773083, creator=18614031015, updator=18614031015), WebsiteProps(id=1308430635954098203, tenantId=1146029695717560320, journalId=null, journalGroupId=null, siteId=1308429426501374870, code=banner, value=null, createTime=1789883773077, updateTime=1789883773077, creator=18614031015, updator=18614031015), WebsiteProps(id=1308430636000235553, tenantId=1146029695717560320, journalId=null, journalGroupId=null, siteId=1308429426501374870, code=grayFlag, value=0, createTime=1789883773088, updateTime=1789883773088, creator=18614031015, updator=18614031015), WebsiteProps(id=1308430635945709594, tenantId=1146029695717560320, journalId=null, journalGroupId=null, siteId=1308429426501374870, code=logo, value=https://castjournals.cast.org.cn/joweb/cdxb/EN/file/pic?fileId=BZ28lIoKdB54MmA0z6K66g==, createTime=1789883773075, updateTime=1789883773075, creator=18614031015, updator=18614031015), WebsiteProps(id=1308430636012818467, tenantId=1146029695717560320, journalId=null, journalGroupId=null, siteId=1308429426501374870, code=minRunFlag, value=0, createTime=1789883773091, updateTime=1789883773091, creator=18614031015, updator=18614031015), WebsiteProps(id=1308430635970875421, tenantId=1146029695717560320, journalId=null, journalGroupId=null, siteId=1308429426501374870, code=picServerUrl, value=https://castjournals.cast.org.cn/joweb/cdxb/EN/file/pic, createTime=1789883773081, updateTime=1789883773081, creator=18614031015, updator=18614031015), WebsiteProps(id=1308430636004429858, tenantId=1146029695717560320, journalId=null, journalGroupId=null, siteId=1308429426501374870, code=silenceFlag, value=0, createTime=1789883773089, updateTime=1789883773089, creator=18614031015, updator=18614031015), WebsiteProps(id=1308430635962486812, tenantId=1146029695717560320, journalId=null, journalGroupId=null, siteId=1308429426501374870, code=staticResourcePath, value=https://castjournals.cast.org.cn/joweb/cast_kjdb_en_623/, createTime=1789883773079, updateTime=1789883773079, creator=18614031015, updator=18614031015), WebsiteProps(id=1308430635987652639, tenantId=1146029695717560320, journalId=null, journalGroupId=null, siteId=1308429426501374870, code=themeColor, value=null, createTime=1789883773085, updateTime=1789883773085, creator=18614031015, updator=18614031015), WebsiteProps(id=1308430635991846944, tenantId=1146029695717560320, journalId=null, journalGroupId=null, siteId=1308429426501374870, code=themeStyle, value=null, createTime=1789883773086, updateTime=1789883773086, creator=18614031015, updator=18614031015)])], journalTitle=草地学报, weixinUrl=null, journalUrl=https://manu40.magtech.com.cn/Jweb_cdxb, iacademicId=null, status=1, seqNo=null, journalTitleEn=Acta Agrestia Sinica, journalPhotoCn=gCdi9sXCX82eLR6S1d4VLg==, journalPhotoEn=4dKqEDfsaGuyf+bpm/3gfw==, journalFirstLetter=C, journalRecommend=null, journalNew=null, journalCollection=null, jcrJf=null, cjcrJf=null, jcrJfStr=null, cjcrJfStr=null, submissionFirstDecision=null, sciSubjectClassification=null, casSubjectClassification=null, citeScore=null, totalCitationFrequency=null, icpCode=null, psCode=null, advertisingLicenseCode=null, copyrightInformation=null, country=null, option=, provinceCode=null, provinceName=null, collectFlag=false, interPubPlatform=, interPubPlatformUrl=null), detailUrlCn=https://castjournals.cast.org.cn/joweb/cdxb/CN/10.11733/j.issn.1007-0435.2026.08.007, detailUrlEn=https://castjournals.cast.org.cn/joweb/cdxb/EN/10.11733/j.issn.1007-0435.2026.08.007, pdfUrlCn=https://castjournals.cast.org.cn/joweb/cdxb/CN/PDF/10.11733/j.issn.1007-0435.2026.08.007, pdfUrlEn=https://castjournals.cast.org.cn/joweb/cdxb/EN/PDF/10.11733/j.issn.1007-0435.2026.08.007, aliStartDate=null, aliEndDate=null, collectionFlag=false, citedCount=null, citedUrl=null, previewStatus=0, delFlag=0, hasFullText=0, orderTime=1786723200000, fullTextJson=null, articleText=null, reference=GAO Y Z,REN J. Advances and prospects in plant root nodule symbiotic nitrogen fixation[J]. Science in China (Series C),2025,55(1):131-145高英志,任健. 植物根瘤共生固氮研究进展与展望[J]. 中国科学(生命科学),2025,55(1):131-145 YANG J,LIU C W,LI X,et al. Advances in the legume–rhizobia symbiosis[J]. Plant Physiology Journal,2023,59(8):1407-1435杨军,刘承武,李霞,等. 豆科植物-微生物共生固氮研究进展[J]. 植物生理学报,2023,59(8):1407-1435 WANG Y N,YANG W,ZUO Y Y,et al. GmYUC2a mediates auxin biosynthesis during root development and nodulation in soybean[J]. Journal of Experimental Botany,2019,70(12):3165-3176 SUN Y,ZHANG J,YANG Z H. Research progress on ecological value and application of Lespedeza plants[J]. Horticulture & Seed,2021,41(8):38-40孙艳,张娟,杨志恒. 胡枝子属植物生态价值及应用研究进展[J]. 园艺与种苗,2021,41(8):38-40 BRUNING B,ROZEMA J. Symbiotic nitrogen fixation in legumes: perspectives for saline agriculture[J]. Environmental and Experimental Botany,2013,92:134-143 TAO J Y,YAN Y X. Application and research progress of GC-MS technology[J]. Yunnan Chemical Technology,2022,49(3):1-3陶静雨,闫玉鑫. GC-MS技术的应用研究进展[J]. 云南化工,2022,49(3):1-3 CAO F,XU N,LIU H J. Application progress of GC-MS in the study of profile-effect of traditional Chinese medicine[J]. Yunnan Chemical Technology,2022,49(12):1-3曹富,徐宁,刘慧娟. GC-MS技术在中药谱效关系研究中的应用进展[J]. 云南化工,2022,49(12):1-3 WANG Y X,LI C,QIU T Y,et al. Response surface optimization of polyphenols extracted from sweetpotato leaves by ultrasonic coupled aqueous two-phase extraction[J]. Journal of Jiangsu Normal University (Natural Science Edition),2018,36(4):44-48王永徐,李臣,邱天越,等. 超声耦合双水相提取甘薯叶多酚的响应面优化[J]. 江苏师范大学学报(自然科学版),2018,36(4):44-48 SAMAVATI V. Polysaccharide extraction from Abelmoschus esculentus: optimization by response surface methodology[J]. Carbohydrate Polymers,2013,95(1):588-597 MIAO Y Y,ZHOU T,SHI S L,et al. Effect of boron on migration and colonization by rhizobia and seedling growth in Medicago sativa[J]. Acta Prataculturae Sinica,2017,26(4):120-133苗阳阳,周彤,师尚礼,等. 硼对根瘤菌在紫花苜蓿体内运移和定殖及对幼苗生长的影响[J]. 草业学报,2017,26(4):120-133 ZHAO L F,DENG Z S,YANG W Q,et al. Diverse rhizobia associated with Sophora alopecuroides grown in different regions of Loess Plateau in China[J]. Systematic and Applied Microbiology,2010,33(8):468-477 GUO Y. Using metabolomics to study the metabolic basis of six leguminous medicinal plants in Inner Mongolia[D]. Harbin:Northeast Forestry University,2020:31-33郭云. 应用代谢组学研究内蒙古6种豆科药用植物的代谢基础[D]. 哈尔滨:东北林业大学,2020:31-33 LIU Q L. Study on secondary metabolites of rhizobia isolated from Sophora alopecuroides[D]. Yangling:Northwest A & F University,2009:26-32刘巧利. 苦马豆根瘤菌次生代谢产物的研究[D]. 杨凌:西北农林科技大学,2009:26-32 ZHAO L. Study on secondary metabolites of endophytes from Astragalus root nodules[D]. Yangling:Northwest A & F University,2009:19-24赵亮. 黄芪根瘤内生菌次生代谢产物研究[D]. 杨凌:西北农林科技大学,2009:19-24 LIU S X. Study on the mechanism of Sinorhizobium meliloti SD101 improving salt tolerance of alfalfa(Medicagosativa.L)[D]. Daqing:Heilongjiang Bayi Agricultural University,2021:23-42刘淑霞. 根瘤菌SD101提高紫花苜蓿耐盐生理和分子机制研究[D]. 大庆:黑龙江八一农垦大学,2021:23-42 WANG W,XU L,FAN Y X,et al. Multivariable comparative analysis of chickpea seed metabolites based on GC-MS[J]. Xinjiang Agricultural Sciences,2023,60(12):2962-2972王威,徐乐,樊艳星,等. 鹰嘴豆种子代谢产物的GC-MS分析[J]. 新疆农业科学,2023,60(12):2962-2972 ZHANG L Y,YU Y B,ZHAO Z Y,et al. Separation and identification of metabolites and metabolic mechanism in two cultivars of mung bean[J]. Food Science,2021,42(16):169-175张丽媛,于英博,赵子莹,等. 不同品种绿豆中代谢产物的分离鉴定及代谢机制分析[J]. 食品科学,2021,42(16):169-175 HU J L. Metabonomics study on soybean treated with extracellular polysaccharides and secreted proteins of Phytophthora sojae[D]. Hefei:Anhui Agricultural University,2019:18-20胡九龙. 大豆疫霉胞外多糖及其外泌蛋白处理下大豆的代谢组学研究[D]. 合肥:安徽农业大学,2019:18-20 WANG L. Metabolomics research of genetically modified rice[D]. Beijing:Beijing University of Chemical Technology,2013:31-35王玲. 转基因水稻的代谢组学研究[D]. 北京:北京化工大学,2013:31-35 ZHOU C N,CHEN L,BIAN Y F,et al. Analysis of ecliptae herba metabolites by GC-MS method[J]. Pharmaceutical Care and Research,2019,19(3):205-209周春娜,陈磊,卞艳芳,等. 墨旱莲代谢产物的GC-MS分析[J]. 药学服务与研究,2019,19(3):205-209 ZHANG J H,YU H Y,DANG J M. Research on inversion model of wheat polysaccharide under high temperature and ultraviolet stress based on dual-spectral technique[J]. Spectroscopy and Spectral Analysis,2023,43(9):2705-2709张郡赫,于海业,党敬民. 基于双光谱技术的高温紫外胁迫下小麦多糖反演模型研究[J]. 光谱学与光谱分析,2023,43(9):2705-2709 REN M. Effects of water stress on tannin and flavonoids in Lespedeza davurica[D]. Taigu:Shanxi Agricultural University,2018:49-53任敏. 水分胁迫对达乌里胡枝子单宁和黄酮类物质的影响[D]. 太谷:山西农业大学,2018:49-53 XU Y Y,WANG Y,YANG W G. Extraction process and antioxidant activity of total flavonoids from alfalfa leaf[J]. Acta Agrestia Sinica,2018,26(3):757-763许英一,王宇,杨伟光. 紫花苜蓿叶总黄酮提取及抗氧化性[J]. 草地学报,2018,26(3):757-763 LIU J. The dissection of ginsenoside metabolic differences controlled by tissue,development and species through metabolomics and transcriptomic approaches[D]. Harbin:Northeast Forestry University,2017:38-45刘佳. 运用代谢组和转录组解析人参皂苷组织和发育特异性的代谢差异[D]. 哈尔滨:东北林业大学,2017:38-45 HU X,LIN N F. Optimization of ultrasonic-assisted extraction of fatty acid from Melilotus officinalis[J]. The Food Industry,2015,36(11):102-105胡雪,林年丰. 响应面法优化黄花草木樨脂肪酸的超声提取工艺[J]. 食品工业,2015,36(11):102-105 DENG G Q,CHEN H Y,LIU R,et al. Application and development of gas chromatography-mass spectrometry technology in food and Chinese herbal medicine testing[J]. Chemical Reagents,2021,43(5):555-562邓高琼,陈亨业,刘瑞,等. 气相色谱-质谱联用技术在食药检测中的应用与发展[J]. 化学试剂,2021,43(5):555-562 DI X Y. Trace level quantification of four cyclic acetals odorous compounds in water by use of headspace SPME followed by GC-MS[J]. Water Purification Technology,2024,43(3):194-203狄欣宜. 顶空固相微萃取-气质联用法测定水中4种痕量环状缩醛类异嗅物质[J]. 净水技术,2024,43(3):194-203 DAI A N,ZHANG L Y,MIN G L,et al. Analysis on the difference of metabolites of naked oats from different produced areas[J]. Food & Machinery,2022,38(3):32-37代安娜,张丽媛,闵广柳,等. 不同产地裸燕麦代谢产物差异分析[J]. 食品与机械,2022,38(3):32-37)
Optimization of Pretreatment Methods for Metabolites of Rhizobia Isolated from Lespedeza davurica Using Box-Behnken Response Surface Methodology
LI Yi-juan, BAI Yu-fang, CHEN Yu-chong, MA Jie, YANG Kai-yuan, CUI Qing-liang, ZHAO Xiang
Affiliations
1 College of Grassland Science, Shanxi Agricultural University, Taigu, Shanxi Province 030800, China; 2 Shanxi Key Laboratory of Grassland Ecological Protection and Native Grass Germplasm Innovation, Taigu, Shanxi Province 030801, China; 3 Youyu Loess Plateau Grassland Ecosystem National Research Station, Youyu, Shanxi Province 037200, China; 4 College of Agricultural Engineering, Shanxi Agricultural University, Taigu, Shanxi Province 030800, China
The symbiont formed by legumes and rhizobia plays a crucial role in the nitrogen cycle, and root exudates significantly affect the nodulation process. The pretreatment and detection methods for metabolites of rhizobia from Lespedeza daurica have not been systematically optimized. In this study, the extraction efficiency of rhizobial metabolites was improved by optimizing pretreatment conditions, including ultrasonic temperature (A), ultrasonic time (B), derivatization temperature (C), and derivatization time (D). Single-factor experiments were used to determine the ranges of detection conditions, followed by a four-factor, three-level Box-Behnken response surface methodology (ultrasonic temperature: 30-40℃ ultrasonic time: 7-11 min derivatization time: 20-100 min derivatization temperature: 50-90℃) for optimization design. A quadratic regression model was established to determine the optimal conditions and verify their reliability. The optimal conditions determined by single-factor experiments were ultrasonic temperature 35℃, ultrasonic time 9 min, derivatization temperature 70℃, and derivatization time 60 min. The response surface model (R2=0.9035) showed that the linear terms A and C, the interaction term CD, and the quadratic terms A2, C2, and D2 had extremely significant effects on the results (P<0.01), while the interaction term AD had a significant effect (P<0.05). After optimization, 145 metabolites were detected, representing an increase of 198.60% compared with the pre-optimization level, with an error of 3.5%. Methodological validation showed that the peak area RSD of the internal standard L-2-chloro-phenylalanine was 8.2% (n=6), and the signal-to-noise ratio (S∶N) was 9.9∶1, which met the requirements for metabolomic detection. This study established a pretreatment method for the detection of metabolites from rhizobia of Lespedeza daurica, providing a technical basis for their extraction and analysis.
Key words
Lespedeza daurica
/
Metabolites of Rhizobia
/
Optimization of pretreatment methods
LI Yi-juan, BAI Yu-fang, CHEN Yu-chong, MA Jie, YANG Kai-yuan, CUI Qing-liang, ZHAO Xiang.
Optimization of Pretreatment Methods for Metabolites of Rhizobia Isolated from Lespedeza davurica Using Box-Behnken Response Surface Methodology[J].
Acta Agrestia Sinica,
2026
, 34
(8)
: 2826
-2836
.
DOI: 10.11733/j.issn.1007-0435.2026.08.007
GAO Y Z,REN J. Advances and prospects in plant root nodule symbiotic nitrogen fixation[J]. Science in China (Series C),2025,55(1):131-145高英志,任健. 植物根瘤共生固氮研究进展与展望[J]. 中国科学(生命科学),2025,55(1):131-145 YANG J,LIU C W,LI X,et al. Advances in the legume–rhizobia symbiosis[J]. Plant Physiology Journal,2023,59(8):1407-1435杨军,刘承武,李霞,等. 豆科植物-微生物共生固氮研究进展[J]. 植物生理学报,2023,59(8):1407-1435 WANG Y N,YANG W,ZUO Y Y,et al. GmYUC2a mediates auxin biosynthesis during root development and nodulation in soybean[J]. Journal of Experimental Botany,2019,70(12):3165-3176 SUN Y,ZHANG J,YANG Z H. Research progress on ecological value and application of Lespedeza plants[J]. Horticulture & Seed,2021,41(8):38-40孙艳,张娟,杨志恒. 胡枝子属植物生态价值及应用研究进展[J]. 园艺与种苗,2021,41(8):38-40 BRUNING B,ROZEMA J. Symbiotic nitrogen fixation in legumes: perspectives for saline agriculture[J]. Environmental and Experimental Botany,2013,92:134-143 TAO J Y,YAN Y X. Application and research progress of GC-MS technology[J]. Yunnan Chemical Technology,2022,49(3):1-3陶静雨,闫玉鑫. GC-MS技术的应用研究进展[J]. 云南化工,2022,49(3):1-3 CAO F,XU N,LIU H J. Application progress of GC-MS in the study of profile-effect of traditional Chinese medicine[J]. Yunnan Chemical Technology,2022,49(12):1-3曹富,徐宁,刘慧娟. GC-MS技术在中药谱效关系研究中的应用进展[J]. 云南化工,2022,49(12):1-3 WANG Y X,LI C,QIU T Y,et al. Response surface optimization of polyphenols extracted from sweetpotato leaves by ultrasonic coupled aqueous two-phase extraction[J]. Journal of Jiangsu Normal University (Natural Science Edition),2018,36(4):44-48王永徐,李臣,邱天越,等. 超声耦合双水相提取甘薯叶多酚的响应面优化[J]. 江苏师范大学学报(自然科学版),2018,36(4):44-48 SAMAVATI V. Polysaccharide extraction from Abelmoschus esculentus: optimization by response surface methodology[J]. Carbohydrate Polymers,2013,95(1):588-597 MIAO Y Y,ZHOU T,SHI S L,et al. Effect of boron on migration and colonization by rhizobia and seedling growth in Medicago sativa[J]. Acta Prataculturae Sinica,2017,26(4):120-133苗阳阳,周彤,师尚礼,等. 硼对根瘤菌在紫花苜蓿体内运移和定殖及对幼苗生长的影响[J]. 草业学报,2017,26(4):120-133 ZHAO L F,DENG Z S,YANG W Q,et al. Diverse rhizobia associated with Sophora alopecuroides grown in different regions of Loess Plateau in China[J]. Systematic and Applied Microbiology,2010,33(8):468-477 GUO Y. Using metabolomics to study the metabolic basis of six leguminous medicinal plants in Inner Mongolia[D]. Harbin:Northeast Forestry University,2020:31-33郭云. 应用代谢组学研究内蒙古6种豆科药用植物的代谢基础[D]. 哈尔滨:东北林业大学,2020:31-33 LIU Q L. Study on secondary metabolites of rhizobia isolated from Sophora alopecuroides[D]. Yangling:Northwest A & F University,2009:26-32刘巧利. 苦马豆根瘤菌次生代谢产物的研究[D]. 杨凌:西北农林科技大学,2009:26-32 ZHAO L. Study on secondary metabolites of endophytes from Astragalus root nodules[D]. Yangling:Northwest A & F University,2009:19-24赵亮. 黄芪根瘤内生菌次生代谢产物研究[D]. 杨凌:西北农林科技大学,2009:19-24 LIU S X. Study on the mechanism of Sinorhizobium meliloti SD101 improving salt tolerance of alfalfa(Medicagosativa.L)[D]. Daqing:Heilongjiang Bayi Agricultural University,2021:23-42刘淑霞. 根瘤菌SD101提高紫花苜蓿耐盐生理和分子机制研究[D]. 大庆:黑龙江八一农垦大学,2021:23-42 WANG W,XU L,FAN Y X,et al. Multivariable comparative analysis of chickpea seed metabolites based on GC-MS[J]. Xinjiang Agricultural Sciences,2023,60(12):2962-2972王威,徐乐,樊艳星,等. 鹰嘴豆种子代谢产物的GC-MS分析[J]. 新疆农业科学,2023,60(12):2962-2972 ZHANG L Y,YU Y B,ZHAO Z Y,et al. Separation and identification of metabolites and metabolic mechanism in two cultivars of mung bean[J]. Food Science,2021,42(16):169-175张丽媛,于英博,赵子莹,等. 不同品种绿豆中代谢产物的分离鉴定及代谢机制分析[J]. 食品科学,2021,42(16):169-175 HU J L. Metabonomics study on soybean treated with extracellular polysaccharides and secreted proteins of Phytophthora sojae[D]. Hefei:Anhui Agricultural University,2019:18-20胡九龙. 大豆疫霉胞外多糖及其外泌蛋白处理下大豆的代谢组学研究[D]. 合肥:安徽农业大学,2019:18-20 WANG L. Metabolomics research of genetically modified rice[D]. Beijing:Beijing University of Chemical Technology,2013:31-35王玲. 转基因水稻的代谢组学研究[D]. 北京:北京化工大学,2013:31-35 ZHOU C N,CHEN L,BIAN Y F,et al. Analysis of ecliptae herba metabolites by GC-MS method[J]. Pharmaceutical Care and Research,2019,19(3):205-209周春娜,陈磊,卞艳芳,等. 墨旱莲代谢产物的GC-MS分析[J]. 药学服务与研究,2019,19(3):205-209 ZHANG J H,YU H Y,DANG J M. Research on inversion model of wheat polysaccharide under high temperature and ultraviolet stress based on dual-spectral technique[J]. Spectroscopy and Spectral Analysis,2023,43(9):2705-2709张郡赫,于海业,党敬民. 基于双光谱技术的高温紫外胁迫下小麦多糖反演模型研究[J]. 光谱学与光谱分析,2023,43(9):2705-2709 REN M. Effects of water stress on tannin and flavonoids in Lespedeza davurica[D]. Taigu:Shanxi Agricultural University,2018:49-53任敏. 水分胁迫对达乌里胡枝子单宁和黄酮类物质的影响[D]. 太谷:山西农业大学,2018:49-53 XU Y Y,WANG Y,YANG W G. Extraction process and antioxidant activity of total flavonoids from alfalfa leaf[J]. Acta Agrestia Sinica,2018,26(3):757-763许英一,王宇,杨伟光. 紫花苜蓿叶总黄酮提取及抗氧化性[J]. 草地学报,2018,26(3):757-763 LIU J. The dissection of ginsenoside metabolic differences controlled by tissue,development and species through metabolomics and transcriptomic approaches[D]. Harbin:Northeast Forestry University,2017:38-45刘佳. 运用代谢组和转录组解析人参皂苷组织和发育特异性的代谢差异[D]. 哈尔滨:东北林业大学,2017:38-45 HU X,LIN N F. Optimization of ultrasonic-assisted extraction of fatty acid from Melilotus officinalis[J]. The Food Industry,2015,36(11):102-105胡雪,林年丰. 响应面法优化黄花草木樨脂肪酸的超声提取工艺[J]. 食品工业,2015,36(11):102-105 DENG G Q,CHEN H Y,LIU R,et al. Application and development of gas chromatography-mass spectrometry technology in food and Chinese herbal medicine testing[J]. Chemical Reagents,2021,43(5):555-562邓高琼,陈亨业,刘瑞,等. 气相色谱-质谱联用技术在食药检测中的应用与发展[J]. 化学试剂,2021,43(5):555-562 DI X Y. Trace level quantification of four cyclic acetals odorous compounds in water by use of headspace SPME followed by GC-MS[J]. Water Purification Technology,2024,43(3):194-203狄欣宜. 顶空固相微萃取-气质联用法测定水中4种痕量环状缩醛类异嗅物质[J]. 净水技术,2024,43(3):194-203 DAI A N,ZHANG L Y,MIN G L,et al. Analysis on the difference of metabolites of naked oats from different produced areas[J]. Food & Machinery,2022,38(3):32-37代安娜,张丽媛,闵广柳,等. 不同产地裸燕麦代谢产物差异分析[J]. 食品与机械,2022,38(3):32-37