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小木虫论坛-学术科研互动平台 » 生物医药区 » 分子生物 » 综合其他 » pGREEN2-0800-LUC载体是否适合农杆菌GV3101
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yudaoqian88

至尊木虫 (知名作家)

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[交流] pGREEN2-0800-LUC载体是否适合农杆菌GV3101

我们将pSOUP辅助质粒转入GV3101，长斑倒是正常的，但无法在10mg/L正常浓度的四环素抗性液体培养基中上生长，不知道是什么原因，一位做过这个实验的同学说需要共转化。今天仔细看了文献，指明可以共转，也可以分两步转化。但在描述合适的菌株时未说明常用的GV3101能否使用，我们在查阅文献方法时，发现多数的研究者利用该载体也都是用的GV3101，特来求助。文献摘取如下：

文献信息如下：
Plant Molecular Biology42:819–832,2000.©2000KluwerAcademicPublishers.PrintedintheNetherlands.819

pGreen: a versatile and fexible binary Ti vector for Agrobacterium-mediated plant transformation

RogerP.Hellens,E.AnneEdwards,NicolaR.Leyland,SamanthaBeanandPhilipM.MullineauxJohnInnesCentre,NorwichResearchPark,Colney,Norwich,NR47UH,UK(authorforcorrespondence;e-mail:hellens@bbsrc.ac.uk;fax:C441603456844)Received8June1999;acceptedinrevisedform30January2000

Keywords: Agrobacterium ,binaryvectors, planttransformation, reportergenes, selectable markergenes, Ti vector

关于转化部分的描述如下：
The pGreen and pSoup plasmids (Figure1) can be used in a mixed electroporation. Inthisinstance, selection for co-transformed Agrobacterium can be achieved using kanamycin-containing medium only, since pGreen can not replicate in Agrobacterium without pSoup being co-resident. Alternatively, electro-competent Agrobacterium containing the pSoup can be generated, by selection for tetracycline resistance, and subsequently re-electroporated with pGreen and selection for kanamycin-resistant colonies: A.tumefaciens strains LBA4404, GV2260, GV3280, AGL-1 and EHA105 and A .rhizogenes strain LBA9402 support  pGreen/pSoup replication.
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yudaoqian88

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我们转过，用的是5

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chemg

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请问psoup可以单转大肠杆菌DH5α吗？转的话tet浓度是多少？ps:我们都是和pgreen共转农杆菌gv3101只加kana抗性，不加tet。希望能帮到你。

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2楼2017-07-20 09:09:59
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3楼: Originally posted by yudaoqian88 at 2017-07-20 10:43:00
我们转过，用的是5

你好，你转化过pSoup的大肠杆菌质粒么？我能在Tet的LB板子上长，但是摇菌摇不起来，能帮忙分析一下吗？
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4楼2018-04-23 16:59:31
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yudaoqian88

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(1) The effector plasmid was constructed by cloning CDS into the vector.
(2) Reporter plasmid, pGreen-LUC, encodes two luciferases: the firefly luciferase controlled by the recombinant E-box promoter and the Renilla luciferase controlled by the constitutive 35S promoter..
(3)  pGreen-LUC reporter plasmid was transformed into Agrobacterium (strain AGL1) together with the helper lasmid pSoup-P19, which also encodes a repressor of cosuppression (Hellens et al., 2005).
(4) Agrobacterium strain containing the reporter Green-LUC was used alone, or mixed with the Agrobacterium strain containing the effector plasmid.
(5) Overnight cultures of Agrobacteria were collected by centrifugation, resuspended in the infiltration buffer (10 mM MES, 150 mM acetosyringone, and 10 mM MgCl2), and incubated at room temperature for 4 h before infiltration. The reporter strain was either incubated alone or as a mixture with the effector strain (at a reporter: effector ratio of 1:1).
(6) Agrobacteria suspension in a 10-mL syringe (without the metal needle) was carefully press-infiltrated manually onto healthy leaves of 21-d-old N. benthamiana (烟草).
(7) Plants were left under continuous white light for 3 d after infiltration, sprayed with luciferin     (1 mM luciferin and 0.01% Triton X-100), and photographed using a charge-coupled device camera (Princeton Instruments).
(8) Leaf samples were collected for the dualluciferase assay using a commercial kit (Promega; DLRreagents). Briefly, leaf discs infected with Agrobacteria were homogenized in 100 mL of passive lysis buffer.
(9) Eight microliters of crude extract was mixed with 40 mL of LUC assay buffer, and the LUC activity was measured using a multimode microplate reader (Berthold; TriStar LB941).
(10) Then, 40 mL of Stop and Glow buffer was added for the measurement of the REN activity.
(11) Multiple biological repeats (n ≥ 3) were performed for each sample.
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