基于Box-Behnken響應面法的烤后煙葉品種分子鑒定技術構建

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中圖分類號：S572.03文獻標識碼：A

文章編號：1007-5119（2025）03-0001-11

Construction of Molecular ldentification Technology for Cured Tobacco Varieties Based on Box-Behnken Response Surface Method

TIAN Zhen1， LI Yuan2，MAO Jingjing]， REN Min2， XIONG Dang'an1， ZHANG Xingwei2， YIN Yudong1，LI Shaopeng'，LIU Guoxing2*

（1. China Tobacco Jiangsu Industrial Co.，Ltd.，Nanjing 21oo19，China; 2. Institute of Tobacco Research， ChineseAcademy ofAgricultural Sciences，Qingdao，China）

Abstract：Toaddress thesevere DNA degradationincured tobacco leaves，whichhinderssubsequent molecular identification experiments，thisstudycomprehensivelyconsideredfactorssuchasDNAconcentrationqualityextractioneficiencycostn safetyformolecuarmarkeraplications.BasedontheSLSmethod，weconductedsingle-factorexperimentsandemploedaBoxBehnken esponse surface model todetermine the optimal DNA extraction protocol forcured tobacco leaves.Sampleamount， centrifugation time，and water bath duration were used as experimental variables， with DNA concentration and the A260/A280 ratio as response values.The results showed that the optimal conditions for DNA extraction from cured tobacco leaves were 20mg of sample， 10 minutes ofcentrifugation，and 15 minutes in water bath. Under these conditions，the DNA concentration was 588.16±27.09ng/μL and the A260/A280 ratio was 1.75±0.03 . These results were consistent with the predictions of Design Expert 11 software， confirming thereliabityoftheoptimizationmethod.MolecularmarkerapplicabilitytestsrevealedthatDNAextractedusingtheotimizedSLS methodieldedclearertargetfragmentpeaks incapilaryelectrophoresisaferPCRamplificationwithSSRandKASPprimers，and distinct genotyping plots wereobtainedbyusing KASPmarkers，demonstrating itsapplicabilityforSSRandKASPmolecularmarker detectioniflue-curedtobacoleaves ofdiferent varieties.The established molecularidentificationsystemcombiningtheoptmized SLSmethodwith13selected SSRprimerpairsefectivelydistinguishedthetestedcuredtobaccovarieties，demonstratingits applicability for cured tobacco cultivar identification.

Keywords： cured tobacco leaf; genomic DNA extraction methods;responsesurface analysis methodology;simple sequence repeat KASP; molecular identification

煙葉生產是煙草行業(yè)發(fā)展的基礎，位于煙草產業(yè)鏈的最前端，是推動煙葉高質量發(fā)展的關鍵。（剩余14948字）