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Current Status and Future Prospects
Published in Stephen P. Slocombe, John R. Benemann, Microalgal Production, 2017
Numerous marker genes have been used for selection of microalgal transformants (Walker et al. 2005). In most cases, these are based on bacterial antibiotic-resistance genes and include markers conferring resistance to zeomycin (the ble gene), paromomycin (aphAV777), hygromycin B (hpt), G418 (npt77), spectinomycin (aadA), chloramphenicol (cat), and nourseothricin (nat). The choice of marker is determined by the particular antibiotic sensitivities of the target species. However, if we consider the regulatory landscape in relation to genetically modified plants, there is concern about the horizontal transfer of such marker genes to pathogenic bacteria. As such, only two commonly used markers are authorized for widespread outdoor application of transgenic crop plants: these are npt77 and hpt. It is, therefore, likely that for commercial applications, the use of other marker genes in transgenic microalgae would not be granted regulatory approval, and indeed the presence of npt77 or hpt in strains to be cultivated outdoors (whether in open ponds or closed photobioreactors) would need to be strongly justified, regulated and closely monitored. One strategy to circumvent the use of bacterial antibiotic-resistance genes is to use dominant alleles of endogenous algal genes as selectable markers (so-called self-cloning). Examples of such markers include alleles of the RPS14 or RPL41 genes that encode 80S ribosomal subunits that confer resistance to emetine and cycloheximide, respectively, or those of the phytoene desaturase gene PDS that confer resistance to norflurazon (Walker et al. 2005).
Agricultural biotechnology
Published in Firdos Alam Khan, Biotechnology Fundamentals, 2018
In order to select only cells that have actually incorporated the new genes, the genes coding for the desired trait are fused to a gene that allows selection of transformed cells, so-called marker genes. The expression of the marker gene enables the transgenic cells to grow in the presence of a selective agent, usually an antibiotic or a herbicide, while cells without the marker gene die. One of the most commonly used markers is the bacterial aminoglycoside-3′ phosphotransferase gene (APH(3′)II), also referred to as neomycin phosphotransferase II (NPTII). This gene codes for an enzyme that inactivates the antibiotics kanamycin, neomycin, and G418 through phosphorylation. In addition to NPTII, a number of other antibiotic resistance genes have been used as selective markers, such as hygromycin phosphotransferase gene conferring resistance to hygromycin. Another group of selective markers is herbicide tolerance genes. Herbicide tolerance has been obtained through the incorporation and expression of a gene that either detoxifies the herbicide in a similar manner as the antibiotic resistance gene products or expresses a product that acts like the herbicide target but is not affected by the herbicide. Herbicide tolerance may not only serve as a trait useful for selection in the development of transgenic plants but also has some commercial interest. Transformation of plant protoplasts, cells, and tissues are usually only useful if they can be regenerated into whole plants. The rate of regeneration varies greatly not only among different species but also between cultivars of the same species. Besides the ability to introduce a gene into the genome of a plant species, regeneration of intact, fertile plants out of transformed cells or tissues is the most limiting step in developing transgenic plants (Figure 6.9).
Plant Biotechnology
Published in Firdos Alam Khan, Biotechnology Fundamentals, 2020
To select only cells that have incorporated the new genes, the genes coding for the desired trait are fused to a gene that allows selection of transformed cells, so-called marker genes. The expression of the marker gene enables the transgenic cells to grow in the presence of a selective agent, usually an antibiotic or an herbicide, while cells without the marker gene die. One of the most commonly used markers is the bacterial aminoglycoside-3′ phosphotransferase gene (APH(3′)II), also referred to as neomycin phosphotransferase II (NPTII). This gene codes for an enzyme that inactivates the antibiotics kanamycin, neomycin, and G418 through phosphorylation. In addition to NPTII, several other antibiotic resistance genes have been used as selective markers, such as hygromycin phosphotransferase gene conferring resistance to hygromycin. Another group of selective markers is herbicide tolerance genes. Herbicide tolerance has been obtained through the incorporation and expression of a gene that either detoxifies the herbicide in a manner similar to that of the antibiotic resistance gene products or a gene that expresses a product that acts like the herbicide target but is not affected by the herbicide. Herbicide tolerance may not only serve as a useful trait for selection in the development of transgenic plants but also has some commercial interest. Transformations of plant protoplasts, cells, and tissues are usually only useful if they can be regenerated into whole plants. The rate of regeneration varies greatly not only among different species but also between cultivars of the same species. Besides the ability to introduce a gene into the genome of a plant species, regeneration of intact, fertile plants out of transformed cells or tissues is the most limiting step in developing transgenic plants (Figure 6.9).
Engineering Trichoderma reesei for the hyperproduction of cellulose induced protein 1 (Cip1) on a sophorose-containing inducer to efficiently saccharify alkali-pretreated corn stover
Published in Preparative Biochemistry & Biotechnology, 2023
Jianghong Li, Yudian Chen, Yushan Gao, Yi Mo, Tingting Long, Bo Yao, Yonghao Li
The T. reesei gene expression vector pPTPDC1 containing both the PDC1 promoter and terminator was constructed in our laboratory, and BsiWI restriction endonuclease sites were added between the promoter and the terminator. pPTPDC1 had previously been used to successfully express the exogenous aabgl1 gene in T. reesei, increasing the β-glucosidase activity of T. reesei by 70-fold.[20] Therefore, after pPTPDC1 was linearized by BsiWI, the Trcip1 gene was ligated between the PDC1 promoter and terminator using the seamless cloning technique to successfully construct a vector overexpressing the Trcip1 gene (pPTPDC1-cip1) (Figure 1A). The T-DNA sequence containing the Trcip1 gene expression cassette and hygromycin B selection marker was integrated into T. reesei Rut C30, and the genomes of the two transformants were extracted. Then, with BsiWI-F and cip1-R as primers, the integration of the Trcip1 gene expression cassette into the genome of T. reesei was demonstrated using PCR (Figure 1B). As verified by DNA sequencing, the expression cassette had no base mutations and could therefore be used for subsequent studies (Figure 1C). The two positive transformants were named T. reesei OE-cip1-1 and OE-cip1-2.