摘要
本试验分别以陇东苜蓿茎尖生长点和下胚轴为外源基因受体材料，进行Lyz-GFP双元基因在苜蓿中的转化和表达的研究。首次在苜蓿中探索性地尝试了农杆菌介导漩涡振荡侵染法转基因新技术，建立了相应的转化程序。分别研究了农杆菌介导的常规转化过程和漩涡振荡茎尖生长点转化过程中若干因素对陇东苜蓿转化苗再生的影响，确定了适宜的农杆菌侵染时间和最适抑制剂浓度，并提出了消除再生苗玻璃化现象的措施。着重对通过漩涡振荡转化法获得的转化苗进行了荧光检测和PCR扩增，初步证明了通过该转化方法Lyz-GFP双元基因已经成功转入陇东苜蓿中，证实了在活体条件下对植物分生组织进行转基因是可行的。
主要研究内容及结果如下：
1．农杆菌介导的陇东苜蓿转Lyz-GFP双元基因的转化体系的建立。以培育5~7d的陇东苜蓿下胚轴为外植体，以传统的农杆菌介导法进行Lyz-GFP双元基因的转化，系统研究了菌液浓度，侵染时间，Kan浓度以及抑菌素浓度对陇东苜蓿再生的影响。结果表明：陇东苜蓿愈伤阶段Kan选择压为60mg/L，分化阶段Kan选择压为70mg/L；适宜的菌液浓度为0.5~0.6；抑菌素选择Cef较好，浓度为250mg/L。
分析和探讨了分化阶段再生苗的玻璃化现象，提出了相应的解决办法。在光照强度为1000～1200Lx条件下，MS培养基中添加2.0mg/L6-BA和0.5mg/LNAA，调整蔗糖浓度为20~25g/L、琼脂浓度为7g/L时可有效降低陇东苜蓿再生苗玻璃化现象。
2．基于漩涡振荡茎尖生长点转化法的陇东苜蓿基因转化体系的建立。以陇东苜蓿2~3d去子叶无菌苗为试验材料，以农杆菌菌液和灭菌石英砂为媒介，进行了Lyz-GFP的双元基因转化。适宜的侵染时间以30min为宜，抑菌素选择Cef，浓度为250mg/L，Kan的筛选浓度为70mg/L，1.0mg/LIBA浓度作为陇东苜蓿诱导生根激素浓度，选择营养土与蛭石1：3的体积配比为移栽陇东苜蓿试管苗的基质配比。
3．转化苗的荧光检测和PCR鉴定。选取相同数量的50株转化苗进行荧光检测，在通过漩涡振荡获得的转化苗中检测到3株植株有明显的荧光蛋白表达；而通过组培获得的转化苗均未检测到荧光表达。对荧光表达植株进行PCR检测，在其中1株中检测到750bp的片段，证明Lyz-GFP双元基因已经成功转入陇东苜蓿。从而为利用茎尖生长点进行基因的转化摸索出一套快速，新型的可行方法。
In this paper, the Alfalfa cultivar LongDong was selected as the study material.Compared to the tissue culture transformation system of LongDong alfalfa, the newtransgenic technology of agrobacterium mediated vortex oscillation infection methodwas invented. By research some factors to affect the regeneration of LongDong alfalfain the transition process, the optimal time of infection and concentration offingbacteria were determined. Lyz-GFP genes was preliminarily proved in the LongDongalfalfa by testing the marker genes green fluorescent protein (GFP) and purpose gene(Lyz) of transformation plant. It confirmed that the technology of plant meristematictissue transgenic in the living conditions is feasible and the stem tip growing pointconversion technologies was tried exploratory in the alfalfa transgenic breedingtechnology.
The main research contents are as follows:
1. The establishment of tissue culture transformation system of transformedLyz-GFP genes LongDong alfalfa. The hypocotyl of growth5~7d LongDong alfalfawas selected as explant to transform Lyz-GFP genes. We have studied the effect ofmany factors on the regeneration of LongDong alfalfa, such as bacteria liquidconcentration, infection, Kan concentration and antibacterial element concentrations.The results show that the optimal Kan concentration for callus stage is60mg/L andthe one for differentiation stage is70mg/L. The optimal bacteria liquid concentrationis0.5~0.6. Cef as antibacterial element is better and the optimal concentration is250mg/L.
In the paper, the Vitrification phenomenon of regeneration plants indifferentiation stage was analyzed and discussed and the solution was put forward.Vitrification phenomenon of LongDong alfalfa was most effective when the followingconditions were met: light intensity is1000~1200Lx, MS culture medium contained2.0mg/L6-BA and0.5mg/LNAA, Sugar concentration is20~25g/L and agarconcentration is7g/L.
2. The establishment of transformation system of agrobacterium mediated vortexoscillation infection method. The sterile LongDong alfalfa of growing2-3d out ofcotyledons was selected as material and agrobacterium-mediated bacterium fluid andsterilization quartz sands were selected as medias when Lyz-GFP genes was transform into alfalfa. The following conditions need to apply:30min was the optimal infectiontime, Cef was selected as antibacterial element and the concentration is250mg/L,screening concentration of Kan is70mg/L. Rooting of LongDong alfalfa needs1.0mg/LIBA, and the tube seedlings of LongDong alfalfa were then transplanted inmatrix of nutritional soil and vermiculite (Volume ratio is1:3).
3. Fluorescence detection and polymerase chain reaction (PCR) identification oftransformational plant. The same number (50transformational plants) were tested byfluorescence detection. Three of these transformational plants by agrobacteriummediated vortex oscillation infection method have fluorescent protein expressionobviously but fluorescence expression was not detected in the transformational plantsdeveloped by the tissue culture.. The three plants were testing by PCR and one of thethree plants has one strip of750bp, so Lyz-GFP genes was transformed into the alfalfaof LongDong successfully. The transgenic technology of stem tip growing pointconversion is a new and fast.
引文
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