Specificity of expression of the GUS reporter gene (uidA) driven by the tobacco ASA2 promoter in soybean plants and tissue cultures

Inaba, Yoshimi; Zhong, Wei Qun; Zhang, Xing‐Hai; Widholm, Jack M.

doi:10.1016/j.jplph.2006.10.009

Cited by 14 publications

(8 citation statements)

References 27 publications

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“…It turned out that in several reports [2,44-52] where promoter strengths were compared, the 5 ′ UTR used were different between the different promoter constructs which could have greatly influenced their conclusions. These reports probably indicate the functionality of a URM (i.e.…”

Section: Discussionmentioning

confidence: 99%

A 28 nt long synthetic 5′UTR (synJ) as an enhancer of transgene expression in dicotyledonous plants

Kanoria

Burma

2012

BMC Biotechnol

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BackgroundA high level of transgene expression is required, in several applications of transgenic technology. While use of strong promoters has been the main focus in such instances, 5′UTRs have also been shown to enhance transgene expression. Here, we present a 28 nt long synthetic 5′UTR (synJ), which enhances gene expression in tobacco and cotton.ResultsThe influence of synJ on transgene expression was studied in callus cultures of cotton and different tissues of transgenic tobacco plants. The study was based on comparing the expression of reporter gene gus and gfp, with and without synJ as its 5′UTR. Mutations in synJ were also analyzed to identify the region important for enhancement. synJ, enhances gene expression by 10 to 50 fold in tobacco and cotton depending upon the tissue studied. This finding is based on the experiments comparing the expression of gus gene, encoding the synJ as 5′UTR under the control of 35S promoter with expression cassettes based on vectors like pBI121 or pRT100. Further, the enhancement was in most cases equivalent to that observed with the viral leader sequences known to enhance translation like Ω and AMV. In case of transformed cotton callus as well as in the roots of tobacco transgenic plants, the up-regulation mediated by synJ was much higher than that observed in the presence of both Ω as well as AMV. The enhancement mediated by synJ was found to be at the post-transcriptional level. The study also demonstrates the importance of a 5′UTR in realizing the full potential of the promoter strength. synJ has been utilized to design four cloning vectors: pGEN01, pBGEN02, pBGEN02-hpt and pBGEN02-ALSdm each of which can be used for cloning the desired transgene and achieving high level of expression in the resulting transgenic plants.ConclusionssynJ, a synthetic 5′UTR, can enhance transgene expression under a strong promoter like 35S as well as under a weak promoter like nos in dicotyledonous plants. synJ can be incorporated as the 5′UTR of transgenes, especially in cases where high levels of expression is required. A set of vectors has also been designed to facilitate this process.

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Section: Discussionmentioning

confidence: 99%

A 28 nt long synthetic 5′UTR (synJ) as an enhancer of transgene expression in dicotyledonous plants

Kanoria

Burma

2012

BMC Biotechnol

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“…The GUS assay was performed according to the procedure described by Inaba et al (2007). The assay buffer contained basic phosphate buffer, anhydrous methanol and X-Gluc.…”

Section: Methodsmentioning

confidence: 99%

Fruit-specific RNAi-mediated suppression of SlNCED1 increases both lycopene and β-carotene contents in tomato fruit

Sun

Yuan

Zhang

et al. 2012

Journal of Experimental Botany

167

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Abscisic acid (ABA) plays important roles during tomato fruit ripening. To study the regulation of carotenoid biosynthesis by ABA, the SlNCED1 gene encoding 9-cis-epoxycarotenoid dioxygenase (NCED), a key enzyme in the ABA biosynthesis, was suppressed in tomato plants by transformation with an RNA interference (RNAi) construct driven by a fruit-specific E8 promoter. ABA accumulation and SlNCED1 transcript levels in the transgenic fruit were down-regulated to between 20–50% of that in control fruit. This significant reduction in NCED activity led to the carbon that normally channels to free ABA as well as the ABA metabolite accumulation during ripening to be partially blocked. Therefore, this ‘backlogged’ carbon transformed into the carotenoid pathway in the RNAi lines resulted in increased assimilation and accumulation of upstream compounds in the pathway, chiefly lycopene and β-carotene. Fruit of all RNAi lines displayed deep red coloration compared with the pink colour of control fruit. The decrease in endogenous ABA in these transgenics resulted in an increase in ethylene, by increasing the transcription of genes related to the synthesis of ethylene during ripening. In conclusion, ABA potentially regulated the degree of pigmentation and carotenoid composition during ripening and could control, at least in part, ethylene production and action in climacteric tomato fruit.

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“…The functional holoenzyme is then assembled in the plastids (Poulsen et al , 1993; Bohlmann et al , 1995; Romero and Roberts, 1996). The ASA β-subunit (ASB1) is an aminotransferase that cleaves glutamine releasing the α-amino group, which is transferred by the ASA α-subunit (ASA1) to chorismate producing anthranilate (Zhang et al , 2001; Inaba et al , 2007). Analyses of the nucleotide sequences coding for ASA α-subunits ( ASA1 ) from rice, Arabidopsis , Ruta graveolens , and tobacco reveal a high degree of sequence homology (Tozawa et al , 2001; Niyogi and Fink, 1992; Bohlmann et al , 1996).…”

Section: Introductionmentioning

confidence: 99%

Increased glutamine in leaves of poplar transgenic with pine GS1a caused greater anthranilate synthetase -subunit (ASA1) transcript and protein abundances: an auxin-related mechanism for enhanced growth in GS transgenics?

Man

Pollmann

Weiler

et al. 2011

Journal of Experimental Botany

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The initial reaction in the pathway leading to the production of indole-3-acetic acid (IAA) in plants is the reaction between chorismate and glutamine to produce anthranilate, catalysed by the enzyme anthranilate synthase (ASA; EC 4.1.3.27). Compared with non-transgenic controls, leaves of transgenic poplar with ectopic expression of the pine cytosolic glutamine synthetase (GS1a; EC 6.3.1.2) produced significantly greater glutamine and significantly enhanced ASA α-subunit (ASA1) transcript and protein (approximately 130% and 120% higher than in the untransformed controls, respectively). Similarly, tobacco leaves fed with 30 mM glutamine and 2 mM chorismate showed enhanced ASA1 transcript and protein (175% and 90% higher than controls, respectively). Furthermore, free IAA was significantly elevated both in leaves of GS1a transgenic poplar and in tobacco leaves fed with 30 mM glutamine and 2 mM chorismate. These results indicated that enhanced cellular glutamine may account for the enhanced growth in GS transgenic poplars through the regulation of auxin biosynthesis.

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Specificity of expression of the GUS reporter gene (uidA) driven by the tobacco ASA2 promoter in soybean plants and tissue cultures

Cited by 14 publications

References 27 publications

A 28 nt long synthetic 5′UTR (synJ) as an enhancer of transgene expression in dicotyledonous plants

A 28 nt long synthetic 5′UTR (synJ) as an enhancer of transgene expression in dicotyledonous plants

Fruit-specific RNAi-mediated suppression of SlNCED1 increases both lycopene and β-carotene contents in tomato fruit

Increased glutamine in leaves of poplar transgenic with pine GS1a caused greater anthranilate synthetase -subunit (ASA1) transcript and protein abundances: an auxin-related mechanism for enhanced growth in GS transgenics?

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