芥菜（Brassica juncea Coss）植株原位真空渗入遗传转化体系的研究

【作者】 金万梅；

【导师】 巩振辉；

【作者基本信息】 西北农林科技大学 ， 蔬菜学， 2001， 硕士

【摘要】 芥菜营养丰富，栽培面积广泛，但在生产上存在着病虫害严重、产量低、品质差等问题。解决的根本途径是培育新品种，常规育种不仅年限长，而且目标性状常常与不良经济性状呈紧密连锁关系。转基因技术能将目标基因直接导入植物染色体组中，育种效率高。国内关子芥菜遗传转化技术还很不成熟，难以适宜育种需求。真空渗入法以其独特的优点受到了广泛的关注和探索。本试验通过对影响转化效果诸因素的研究试图建立芥菜植株原位真空渗入高效遗传转化体系，为该法的广泛应用提供理论依据和方法技术。 本项研究于1999年6月至2001年5月，以芥菜雪里凝和园叶芥为材料，采用人工低温处理和春秋播期试验以及对影响原位真空渗入遗传转化效果的真空渗入时间、菌液浓度、渗入培养基、芥菜生长发育时期、组织器官和农杆菌菌株进行处理试验，建立了芥菜原位真空渗入遗传转化体系，获得了以下主要结果： 1．雪里散和圆叶芥芥菜种子经 50C温烫 10min，23oC浸种 2－6h，催芽 20h，或 23t浸种 5 h不催芽，置0－4’C处理25d 圆叶芥9月20日、9月30日和2月27日，雪里数9月30日和2月27日露地播种，均易形成芥菜植株原位真空渗入遗传转化适宜苗态。 2．通过对影响转化效果的诸因素的研究，建立了芥菜植株原位真空渗入高效遗传转化方法：真空渗入时间为Zmin／间歇30s／30s或Zmin／间歇1．smin／30s；菌液浓度OD。为0．8－l二，渗入培养基为 l／ZMS无机十B。有机；菌株采用强感染型的；在蕾期对花蕾进行真空渗入。 3．初步建立一种鉴定转基因植株的方法--生长点点滴筛选法，该法筛选雪里敦和圆叶芥的最佳潮霉素浓度为 800mg／L，筛选时间自接种潮霉素后 10d开始。筛选转化体的标准：真叶长出，叶色显绿，生长正常。由于其不需要培养基，无污染，节省药品，筛选出的转化体移栽成活率高，具有一定的应用前景。 4．建立了培养基筛选芥菜转基因植株的潮霉素最佳浓度和筛选最佳时期。25mg／L为芥菜雪里票和圆叶芥潮霉素筛选的最佳浓度；筛选的最佳时间从16日龄开始。鉴别转化体的标准：在潮霉素浓度为 25m叭的筛选培养基上，16日龄以上的雪里燕和圆叶芥植株生长正常，叶片现绿，有侧根发出或真叶长出或既有侧根又有真叶出现。 5．参考微量DNA提取方法，成功地提取了用于PCR扩增的芥菜DNA，并建立了PCR扩增反应体系。 6．获得了转基因植株。本试验经潮霉素筛选获抗性苗386棵，随机取样六棵，对其提取基因组DNA，进行PCR分子检测，均有条带扩出，证明外源基因己整合在芥菜基因组DNA上。更多还原

【Abstract】 MUSnd Hicajbecea Coss．) is widely planed and it’s a nutritive vegetable． But there existserious plant diseases and insect Pests, Low yield and bad quality Only resolving method is throughcultUring new cultivatOL Conventional breeding demands longer time and eqet gene aPpere too late, WhilethrOUgh thesgenetic technology W gene is inserted plant cell, So breeding efficiency is linproved raPidlyHowever the genetic transformation technology in mustards had not gained breakthrugh． The vacuuminfiltraion genetic bosformation with unique advantages is aPplied and stUdied． Thrugh StUdying on theeffeCts of the tusformatic facors in order to establish in sitU vacuum infiboion genehc tranSformationsystem of mUSeds and provide the theOreicaI basis． Main conclusions are as follow:l．After MtJStards seeds at 50C hot watr were warmedl0min, at23C soaked for 24h, at 23oC sProuted for20h or at 50C hot waer were wanned l0min, at 23C soaked 5h, then aMC treated for 25days, then plantedin soil, they were eaSy to form optiInum plant ShaPe for vacuum whhatio ~c taformallon during lOngday light． Cv YUanyejie seeds being sowed in oPen field on 20 SePtember, on 30 SePtember and on l7 Febmpand CvXuelthong seeds being sowed in open fieId on 30 SePtember and on 27 FebrUary are eaSy to formoPtimum plat shaPe for in situ vacuum infiIthaon genetic tranSformation．2． Through studying on the factors of the effects on genetic traflsformation． On Mustards plant in sittIvacuum infiltration high efficiency genetic transformation was established． The vacuum infiItration timeis 2minj intermission30s/30s or 2minjintermissiom 1．5minj30s． Agrobacterium concentration is OD00o0．8． Infiltration medium is l/2MS inorganic composition and B5 organic composition． In bud stage thebuds are treated by the vacuum infiltTation．3． Primarily a method of identifitalion of triformatic plant was consmictedWwth point selection． Onscreening CvXuelthong and YUanyeie seeds, opdritim conCetaion is 800 mgh and oPtimurn time ofscreening is l0 days afle being inoculated． It does not reqube medium, not POllute, save medicinamen andgain high survive． The hygromycin resiStance seedling: leaf buds appear become gree and grOW normally4． On screening CvXuelihong and Yuanyejie seedsthrough medium with hygromycin, optimumconcentration is 25 mg/L and optimum time of screening is l6 days5． The genome DNA was ertted bough microbial etating method and ethelished PCR reaction system6． Transgenetic plants were obtained． There were 386 Hyg resistance seedlings, and at random sevenseedlings were selected among them, and then their leaves were eXtr8cted into DNA and made PCRidentification． It proved that the foreign-gene was collocated into genome．更多还原