Genetic Transformation of The Green Algae Micractinium tetrahymenae by Agrobacterium Mediated transformation;Genetic Transformation of The Green Algae Micractinium tetrahymenae by Agrobacterium Mediated transformation

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题名:	Genetic Transformation of The Green Algae Micractinium tetrahymenae by Agrobacterium Mediated transformation;Genetic Transformation of The Green Algae Micractinium tetrahymenae by Agrobacterium Mediated transformation
作者:	孟里凡;munir, ivan mambaul
贡献者:	生命科學系
关键词:	Agrobacterium Mediated Transformation;The Green algae;Genetic modification;Micractinium tetrahymenae;Biomass;Agrobacterium Mediated Transformation;The Green algae;Genetic modification;Micractinium tetrahymenae;Biomass
日期:	2021-07-30
上传时间:	2021-12-07 11:23:56 (UTC+8)
出版者:	國立中央大學
摘要:	綠藻被視為是一個生產生物質的理想平台，而生物質可以運用在人類及動物的食物，以及進一步生產生質能源。基因改造已經變成是一項可以加速生物質生產之強而有力的工具，並且可以降低生產時的花費，使得整體生產更加經濟實惠。然而，一直以來綠藻的基因改造總是面對許多困難，例如:低的轉型成功率、不同數目的轉基因拷貝數以及不穩定的基因表現。在許多轉殖外來基因的轉型方法中，農桿菌 (Agrobacterium sp.) 調控的轉型方法最為常用。雖然這項技術在一些藻類中可以作用，包括: Chlamydomonas reinhardtii, Haematococcus pluvialis 以及 Chorella vulgaris，但是轉型的效率會因不同的物種而有所差異。Micractinium tetrahymenae，一種會與四膜蟲形成內共生的綠藻，具有作為生產平台很好的前瞻，因為其具有產生大量的生物質的可能性。在這項研究中，我們首次成功的將帶有帶有潮黴素以及綠螢光蛋白T-DNA的Agrobacterium tumefaciens，轉型Micractinium tetrahymenae 的基因體中。轉型的方法是基於潮黴素抗藥性的表現型，我們也更進一步地提供PCR (聚合?連鎖反應) 以及綠色螢光強度的差異作為轉型成功的證據。;The green algae are considered as an ideal platform for biomass production which can be used for human food, animal feed and biofuels. Genetic modification has become a robust approach to speed up the development of biomass productions and reduce the production cost, making the production economically feasible. The main issues for the genetic modification of green algae are the low transformation efficiency, varied copy numbers of transgene integration and instability of gene expression. Among the transformation methods for delivering exogenous DNA, Agrobacterium-mediated transformation is the most commonly used. Although this technique has been shown to work with a few algae, including Chlamydomonas reinhardtii, Haematococcus pluvialis and Chorella vulgaris, its working efficiency varies depending on the species. The green algae, Micractinium tetrahymenae, has a great prospect as a production platform due to its potential ability to produce a large amount of biomass and the first green algae representative of endosymbiosis found in Tetrahymena utriculariae. In this study, we report the first successful transfer of Agrobacterium tumefaciens T-DNA carrying the genes coding for a drug (hygromycin) resistant marker and green fluorescent protein (GFP) to the nuclear genome of Micractinium tetrahymenae. The transformation protocol was based on the hygromycin resistance phenotype. We further provide transformation evidence by PCR and measuring the green fluorescence intensity in the transformed cells.
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