基于Box-Behnken响应面法的烤后烟叶品种分子鉴定技术构建

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中图分类号：S572.03文献标识码：A

文章编号：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

烟叶生产是烟草行业发展的基础，位于烟草产业链的最前端，是推动烟叶高质量发展的关键。（剩余14948字）