Nuclear transformation of the diatom Phaeodactylum tricornutum using PCR-amplified DNA fragments by microparticle bombardment
Nuclear transformation of the diatom Phaeodactylum tricornutum using PCR-amplified DNA fragments by microparticle bombardment
复制标题
DOI：
10.1016/j.margen.2015.12.004
复制
发表时间：
2016-02-01
期刊：
影响因子：
1.9
通讯作者：
Adachi, Masao
中科院分区：
文献类型：
--
作者：
Kira, Nozomu;Ohnishi, Kohei;Adachi, Masao
我们已经发展了一种利用聚合酶链反应（PCR）扩增的DNA片段通过微粒轰击转化海洋硅藻的方法。我们构建了一个环状载体（约5000 bp），其中包含一个来自三角褐指藻的fcpA启动子，一个来自梭形棘球绦虫的抗虫基因和终止子（一个“基因盒”）。然后从环状质粒中PCR扩增不同长度的线性载体（+0-+1000线性载体）。在一式三份实验中获得用线性载体转染的三角褐指藻的转化体。使用含有基因盒和在基因盒两端的0、50和500 bp的额外DNA区域的PCR扩增的短线性载体（+0-+500线性载体）的转化效率彼此之间或与+1000线性载体的效率没有显著差异。使用线性载体的转化效率低于使用圆形载体的转化效率，但没有显著差异。测定含有基因盒的整个区域的转化体的数目与使用各种长度的线性载体转染的转化体的数目的比率。DNA片段的延伸（>= 50 bp）对于将基因盒的整个区域引入基因组DNA是有效的。在使用不同量的+50线性载体（37.5-300 fmol/shot）时，我们观察到使用37.5 fmol（52.2 ng）/shot的线性载体的转化效率与使用300 fmol线性载体获得的转化效率没有显著差异。考虑到环状质粒的量（1 μ g =约100 μ g），设定300 fmol的量。300 fmol）和37.5 fmol的量被设定为在一个含有50 μ l PCR混合物的PCR管中快速和容易地制备三次转化实验所需的约500 ng线性短载体。使用Southern印迹分析确定将短线性载体的基因盒以及全长线性载体（+1000线性载体）的基因盒整合到染色体DNA中。短线性载体倾向于导致比超螺旋质粒更少的插入数目。这种简单和省时的转化方法，使用微粒子轰击与PCR扩增的DNA片段允许的功能分析的硅藻特异性基因和硅藻菌株的发展，可用于进一步的生物技术应用。(C)2015爱思唯尔B. V.保留所有权利。
We have developed a method for marine diatom transformation by microparticle bombardment using polymerase chain reaction (PCR)-amplified DNA fragments. We constructed a circular vector (approximately 5000 bp) containing an fcpA promoter from Phaeodactylum tricornutum, antibiotic-resistance genes and terminator from Cylindrotheca fusiformis (a "gene cassette"). Then the various lengths of linear vectors (+0-+1000 linear vectors) were then PCR-amplified from the circular plasmid. The transformants of P. tricornutum transfected with the linear vectors were obtained in the triplicate experiments. Transformation efficiencies using PCR-amplified short linear vectors containing the gene cassette and additional DNA regions of 0, 50, and 500 bp at both ends of the gene cassette (+0-+500 linear vectors) did not significantly differ from one another or from the efficiency of the +1000 linear vector. Transformation efficiencies using the linear vectors were lower than that using the circular vector, but were not significantly different. The ratios of the number of transformants containing the whole region of the gene cassette to those of transformants transfected using linear vectors of various lengths were determined. An extension (>= 50 bp) of DNA fragments was effective for introducing the whole region of the gene cassette into the genomic DNA. In using various amounts of the +50 linear vector (37.5-300 fmol/shot), we observed that transformation efficiencies using 37.5 fmol (52.2 ng)/shot of the linear vector were not significantly different from those obtained using 300 fmol of the linear vector. The 300 fmol quantity was set considering the quantity of the circular plasmid (1 mu g = approx. 300 fmol) and the 37.5 fmol quantity was set for quick and easy preparation of approximately 500 ng of the linear short vector needed for triplicate transformation experiments in one PCR tube containing 50 mu l of PCR cocktail. Integrating the gene cassette of the short linear vectors as well as that of the full length of the linear vector (+ 1000 linear vector) into the chromosomal DNA was determined using Southern blot analysis. The short linear vectors tended to result in smaller numbers of insertions than those of the supercoiled plasmid. This simple and timesaving transformation method using microparticle bombardment with PCR-amplified DNA fragments permitted both functional analysis of diatom-specific genes and development of diatom strains useful for further biotechnological applications. (C) 2015 Elsevier B.V. All rights reserved.