I want to expression GFP in lettuce. I checked wild type plant by fluorsecent microscope. I saw bright green in vessel elements (attached file).
what may be the reason? What should I do next?
Does any one also have such experience?
Thanks a lot.
GFP fluoresces at a similar wave length to lignin. I suspect it is the lignin that you are observing. Plant vessels are cells with highly thickened cell walls and part of secondary cell wall formation is the deposition of large amounts of lignin. Lignin helps to waterproof these cells, which carry water from the roots to the extremities of the plant. I think if you fine tune the wavelength you should be able to remove the background expression but for plant work I have often seen YFP, CFP and mCherry being used instead.
Try to use RFP or any other not green protein.
I have no idea about the source of that fluorescence...
GFP fluoresces at a similar wave length to lignin. I suspect it is the lignin that you are observing. Plant vessels are cells with highly thickened cell walls and part of secondary cell wall formation is the deposition of large amounts of lignin. Lignin helps to waterproof these cells, which carry water from the roots to the extremities of the plant. I think if you fine tune the wavelength you should be able to remove the background expression but for plant work I have often seen YFP, CFP and mCherry being used instead.
I think your source is somehow contaminated. Did you replicated this experiment? Collect another wild type plant. Which part of the plant you took?
Regards
NZ
Thank you all, especially Nicky...
Dear Nayem, I checked 10 different plants leaves, but i saw same result.
If we check GFP expression at callus level, i think the problem mentioned can be avoided, what do you think dear Mostafa?
هدف شما از بیان جی اف پی چیست ؟ بررسی بیان یک ژن یا تعیین محل استقرار یک پروتئین؟
در مرحله اول می توانید از بافت بدون آوند استفاده کنید. یک مسئله در که در ارتباط با مشاهده ۀ جی اف پی وجود دارد فلور سانس قرمزکلروفیل در حضور یو وی است که در این عکس مشاهده نمی شود به همین دلیل است که معمولا برای مشاهده از بافت های فاقد کلروفیل مانند اپیدرم استفاده می شود. آوند ها معمولا نوری به رنگ آبی دارند که با رنگ ناشی از جی اف پی کمی تفاوت دارد و شما پس از مشاهدات متعدد و مقایسه با گیاه تراریخت و جی اف پی واقعی می توانید آن را تشخیص دهید . برای امتحان می توانید یک تار پنبه را با میکروسکوپ و نور یو وی مشاهده کنید و این فلورسانس را بدون تراریخت بودن تار پنبه مشاهده نمایید که که پاسخ قبلی مبنی بر فلورسانس لیگنین را تایید می نماید. امیدوارم که نتایج خوبی بگیرید
Most problable answer - lignin. Try plants of younger age and or another cultivar.
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