Transient chimeric gene expression in pollen of five conifer species following microparticle bombardment

1994 ◽  
Vol 24 (12) ◽  
pp. 2417-2423 ◽  
Author(s):  
Irene Hay ◽  
Denis Lachance ◽  
Patrick Von Aderkas ◽  
Pierre J. Charest
Keyword(s):  
Gene Expression ◽  
Black Spruce ◽  
Lodgepole Pine ◽  
Western Hemlock ◽  
35S Promoter ◽  
Specific Gene ◽  
Gus Gene ◽  
Npt Ii ◽  
Gus Gene Expression ◽  
Npt Ii Gene

Mature pollen of lodgepole pine (Pinusconcorta Dougl.), yellow cypress (Chamaecyparisnootkatensis (D. Don) Spach), western hemlock (Tsugaheterophylla (Raf.) Sarg.), jack pine (Pinusbanksiana Lamb.), and black spruce (Piceamariana (Mill.) B.S.P.) was bombarded with gold particles coated with four different plasmid constructions, pRT99GUS, pBM113Kp, pAct1-D, and pGA984, using the biolistic PDS-1000/He device. A protocol was devised for efficient gene transfer and gene expression assay in pollen. False positive results for expression of the β-glucuronidase (GUS) gene assayed with the substrate X-glucuronide were observed with pollen of yellow cypress, western hemlock, and lodgepole pine. The highest levels of transient GUS gene expression were obtained with plasmid pBM113Kp, which carried the GUS gene under the control of the wheat abscisic acid inducible early methionine promoter. The plasmids pRT99GUS (35S promoter) and pAct1-D (rice actin promoter) yielded similar intermediate levels of transient GUS gene expression. The pollen-specific promoter of the α-tubulin gene from Arabidopsisthaliana (pGA984) yielded the lowest levels of gene expression in pollen. Of the four species, yellow cypress showed the lowest levels of transient GUS gene expression and black spruce yielded the highest levels. The neomycin phosphotransferase II (NPT II) gene was also tested as a reporter gene for pollen transformation and was easily assayed via ELISA. The fusion gene between NPT II and GUS genes was detected at a lower level than the nonfused NPT II gene when under the control of the same 35S promoter. The method devised here could be used for the study of tissue-specific gene expression in conifer pollen.

scholarly journals Evaluation of Plant-Derived Promoters for Constitutive and Tissue-Specific Gene Expression in Potato

Plants ◽  
2020 ◽  
Vol 9 (11) ◽  
pp. 1520
Author(s):  
Dmitry Miroshnichenko ◽  
Aleksey Firsov ◽  
Vadim Timerbaev ◽  
Oleg Kozlov ◽  
Anna Klementyeva ◽  
...  
Keyword(s):  
Gene Expression ◽  
Specific Activity ◽  
Expression Patterns ◽  
Camv 35S Promoter ◽  
35S Promoter ◽  
Specific Gene ◽  
Transcriptional Level ◽  
Promoter Strength ◽  
Tissue Specific ◽  
Camv 35S

Various plant-derived promoters can be used to regulate ectopic gene expression in potato. In the present study, four promoters derived from the potato genome have been characterized by the expression of identical cassettes carrying the fusion with the reporter β-glucuronidase (gusA) gene. The strengths of StUbi, StGBSS, StPat, and StLhca3 promoters were compared with the conventional constitutive CaMV 35S promoter in various organs (leaves, stems, roots, and tubers) of greenhouse-grown plants. The final amount of gene product was determined at the post-transcriptional level using histochemical analysis, fluorometric measurements, and Western blot analysis. The promoter strength comparison demonstrated that the StUbi promoter generally provided a higher level of constitutive β-glucuronidase accumulation than the viral CaMV 35S promoter. Although the StLhca3 promoter was predominantly expressed in a green tissue-specific manner (leaves and stems) while StGBSS and StPat mainly provided tuber-specific activity, a “promoter leakage” was also found. However, the degree of unspecific activity depended on the particular transgenic line and tissue. According to fluorometric data, the functional activity of promoters in leaves could be arranged as follows: StLhca3 > StUbi > CaMV 35S > StPat > StGBSS (from highest to lowest). In tubers, the higher expression was detected in transgenic plants expressing StPat-gusA fusion construct, and the strength order was as follows: StPat > StGBSS > StUbi > CaMV 35S > StLhca3. The observed differences between expression patterns are discussed considering the benefits and limitations for the usage of each promoter to regulate the expression of genes in a particular potato tissue.

Studies of Cytokinin Action and Metabolism Using Tobacco Plants Expressing either the ipt or the GUS Gene Controlled by a Chalcone Synthase Promoter. IIipt and GUS Gene Expression, Cytokinin Levels and Metabolism

1997 ◽  
Vol 24 (5) ◽  
pp. 673 ◽  
Author(s):  
Jian Wang ◽  
D. S. Letham ◽  
Edwina Cornish ◽  
K. Wei ◽  
C. H. Hocart ◽  
...  
Keyword(s):  
Gene Expression ◽  
Chalcone Synthase ◽  
Hormonal Control ◽  
Mature Leaf ◽  
Cytokinin Oxidase ◽  
Endogenous Cytokinin ◽  
Gus Gene ◽  
Ipt Gene ◽  
Cytokinin Level ◽  
Gus Gene Expression

The expression of GUS and ipt genes under control of a chalcone synthase (chs) promoter (PCHS) has been determined in tobacco (Nicotiana tabacum L.) plants and related to the development of plants expressing the chimaeric PCHS -ipt gene. GUS gene expression, which served as a model for the expression of the ipt gene, was highest in the internal phloem tissue of stems, in mature leaf laminae and in the upper part of corollas when fully open. Expression of the PCHS -ipt gene was assessed by quantifying the cytokinins produced, by determining incorporation of [3H]adenine into cytokinins and by quantifying ipt mRNA. Results from these studies were in general agreement with those based on expression of the PCHS -GUS gene. The chs promoter controlled expression of the ipt gene with some degree of tissue and temporal specificity. Expression of the ipt gene markedly elevated the cytokinin level in mature leaf laminae and the upper stems of flowering plants. The former was associated with retardation of leaf senescence and increased rates of transpiration due to changes in number, size and aperture of stomata, while the latter was associated with development of lateral shoots. In shoot tip cultures, 2-fold elevations in endogenous cytokinin level caused clear changes in development and this is discussed in relation to current concepts concerning the hormonal control of plant development. Using the transgenic tobacco tissues, it was shown that cis-zeatin is a substrate for cytokinin oxidase, that cis-zeatin is not converted to trans-zeatin in these tissues and that the endogenous cytokinin level influences the level of cytokinin oxidase activity in tissue and the rate of degradation of exogenous zeatin riboside to adenosine.

Transient GUS gene expression in Brassica napus electroporated microspores

Plant Science ◽  
1993 ◽  
Vol 93 (1-2) ◽  
pp. 177-184 ◽  
Author(s):  
Marie-Françoise Jardinaud ◽  
André Souvré ◽  
Gilbert Alibert
Keyword(s):  
Gene Expression ◽  
Brassica Napus ◽  
Gus Gene ◽  
Gus Gene Expression

scholarly journals STUDI PERBANDINGAN METODE TRANSFORMASI DNA MENGGUNAKAN VEKTOR Agrobacterium tumefaciens PADA TANAMAN TEBU (Sacharum hybrid)

2007 ◽  
Vol 13 (1) ◽  
pp. 39-44
Author(s):  
Sri Setyati ◽  
Purnama Oktaviandari ◽  
Muhammad Hazmi ◽  
Bambang Sugiharto
Keyword(s):  
Gene Expression ◽  
Embryogenic Callus ◽  
Multiple Shoots ◽  
Suspension Cultures ◽  
Gus Gene ◽  
In Vitro Plants ◽  
Histochemical Observation ◽  
Selection Medium ◽  
Gus Gene Expression

In order to compare transient expression of gus gene driven by CaMV 35S and rice ubiquitin RUBQ2 promoters, a DNA transformation was conducted using embryogenic callus and suspension cultures of sugarcane. The transient gus expression was observed by histochemical staining method. The histochemical observation of GUS activity after co-cultivation showed that RUBQ2 promoter produced high level of clear blue spots both in embryogenic callus and suspension cultures, while the CaMV35S promoter was not detected. The suspension cultures slightly increased transient gus gene expression compared to embryogenic callus. However, the histochemical analysis of regenerated putative transformant plants after 5 successive cycles on the selection medium showed no blue spots of gus gene expression. PCR amplification of DNA for CaMV35 or nptII in putative transformant plants confirmed that there was no integration of the transformed gene in the genome DNA. The results suggested a possibility of somaclonal variation with callus propagation, thus did not produce transformed plants. To avoid the somaclonal variation, the transformation was conducted using in vitro plants and multiple shoots without intervening callus phase. Histochemical observation of infected materials after co-cultivation showed that almost all of the infected materials partially exhibited blue color in the basal region. In case of in vitro plants, they rapidly grow and multiplied in the selection medium, thus the method provided an excellent system for the transformation in sugarcane. The results suggest that in vitro plants as well as multiple shoots need further investigation to be used as target tissues for Agrobacteriummediated transformation in sugarcane.

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