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A built-in strategy for containment of transgenic plants: creation of selectively terminable transgenic rice.

Lin C, Fang J, Xu X, Zhao T, Cheng J, Tu J, Ye G, Shen Z - PLoS ONE (2008)

Bottom Line: Simple and reliable containment strategies for transgenes are highly desirable.We generated transgenic rice plants by this method using a new glyphosate resistant 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene from Pesudomonas putida as the gene of interest, and demonstrated that these transgenic rice plants were highly sensitive to bentazon but tolerant to glyphosate, which is exactly the opposite of conventional rice.Therefore, this method of creating transgenic rice constitutes a novel strategy of transgene containment, which appears simple, reliable and inexpensive for implementation.

View Article: PubMed Central - PubMed

Affiliation: Institute of Insect Sciences and State Key Laboratory of Rice Biology, Zhejiang University, Hangzhou, China.

ABSTRACT
Plant transgenic technology has been widely utilized for engineering crops for trait improvements and for production of high value proteins such as pharmaceuticals. However, the unintended spreading of commercial transgenic crops by pollination and seed dispersal is a major concern for environmental and food safety. Simple and reliable containment strategies for transgenes are highly desirable. Here we report a novel method for creating selectively terminable transgenic rice. In this method, the gene(s) of interest is tagged with a RNA interference cassette, which specifically suppresses the expression of the bentazon detoxification enzyme CYP81A6 and thus renders transgenic rice to be sensitive to bentazon, a herbicide used for rice weed control. We generated transgenic rice plants by this method using a new glyphosate resistant 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene from Pesudomonas putida as the gene of interest, and demonstrated that these transgenic rice plants were highly sensitive to bentazon but tolerant to glyphosate, which is exactly the opposite of conventional rice. Field trial of these transgenic rice plants further confirmed that they can be selectively killed at 100% by one spray of bentazon at a regular dose used for conventional rice weed control. Furthermore, we found that the terminable transgenic rice created in this study shows no difference in growth, development and yield compared to its non-transgenic control. Therefore, this method of creating transgenic rice constitutes a novel strategy of transgene containment, which appears simple, reliable and inexpensive for implementation.

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RT-PCR analysis of CYP81A6 mRNA abundance in transgenic and non-transgenic rice.The rice Actin gene was used as the control. M, DNA size maker; CK, non-transgenic rice; R450-2, R450-5, R450-6 and R450-7 are independent transgenic events sensitive to bentazon.
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pone-0001818-g004: RT-PCR analysis of CYP81A6 mRNA abundance in transgenic and non-transgenic rice.The rice Actin gene was used as the control. M, DNA size maker; CK, non-transgenic rice; R450-2, R450-5, R450-6 and R450-7 are independent transgenic events sensitive to bentazon.

Mentions: RT-PCR was used to estimate the relative abundance of the CYP81A6 mRNA in transgenic rice and non-transgenic rice. The total RNAs were isolated from the non-transgenic control plants and the transgenic plants of each event of R450-2, R450-5, R450-6, and R450-7, respectively. The total RNAs were used as the templates for the one-step RT-PCR analysis of the rice CYP81A6 gene and Actin gene. We found that the amount of the 310bp CYP81A6 RT-PCR product was greatly reduced from the transgenic plants compared to that from the non-transgenic control plants, while the amount of the 440bp Actin RT-PCR product was roughly equal among the transgenic and non-transgenic plants (Fig. 4). This result suggested that the CYP81A6 mRNA in these transgenic plants was significantly suppressed. This is in agreement with our assumption that the RNA interference cassette introduced in tandem with the gene of interest in these transgenic rice plants is responsible for their sensitivity to bentazon.


A built-in strategy for containment of transgenic plants: creation of selectively terminable transgenic rice.

Lin C, Fang J, Xu X, Zhao T, Cheng J, Tu J, Ye G, Shen Z - PLoS ONE (2008)

RT-PCR analysis of CYP81A6 mRNA abundance in transgenic and non-transgenic rice.The rice Actin gene was used as the control. M, DNA size maker; CK, non-transgenic rice; R450-2, R450-5, R450-6 and R450-7 are independent transgenic events sensitive to bentazon.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2262152&req=5

pone-0001818-g004: RT-PCR analysis of CYP81A6 mRNA abundance in transgenic and non-transgenic rice.The rice Actin gene was used as the control. M, DNA size maker; CK, non-transgenic rice; R450-2, R450-5, R450-6 and R450-7 are independent transgenic events sensitive to bentazon.
Mentions: RT-PCR was used to estimate the relative abundance of the CYP81A6 mRNA in transgenic rice and non-transgenic rice. The total RNAs were isolated from the non-transgenic control plants and the transgenic plants of each event of R450-2, R450-5, R450-6, and R450-7, respectively. The total RNAs were used as the templates for the one-step RT-PCR analysis of the rice CYP81A6 gene and Actin gene. We found that the amount of the 310bp CYP81A6 RT-PCR product was greatly reduced from the transgenic plants compared to that from the non-transgenic control plants, while the amount of the 440bp Actin RT-PCR product was roughly equal among the transgenic and non-transgenic plants (Fig. 4). This result suggested that the CYP81A6 mRNA in these transgenic plants was significantly suppressed. This is in agreement with our assumption that the RNA interference cassette introduced in tandem with the gene of interest in these transgenic rice plants is responsible for their sensitivity to bentazon.

Bottom Line: Simple and reliable containment strategies for transgenes are highly desirable.We generated transgenic rice plants by this method using a new glyphosate resistant 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene from Pesudomonas putida as the gene of interest, and demonstrated that these transgenic rice plants were highly sensitive to bentazon but tolerant to glyphosate, which is exactly the opposite of conventional rice.Therefore, this method of creating transgenic rice constitutes a novel strategy of transgene containment, which appears simple, reliable and inexpensive for implementation.

View Article: PubMed Central - PubMed

Affiliation: Institute of Insect Sciences and State Key Laboratory of Rice Biology, Zhejiang University, Hangzhou, China.

ABSTRACT
Plant transgenic technology has been widely utilized for engineering crops for trait improvements and for production of high value proteins such as pharmaceuticals. However, the unintended spreading of commercial transgenic crops by pollination and seed dispersal is a major concern for environmental and food safety. Simple and reliable containment strategies for transgenes are highly desirable. Here we report a novel method for creating selectively terminable transgenic rice. In this method, the gene(s) of interest is tagged with a RNA interference cassette, which specifically suppresses the expression of the bentazon detoxification enzyme CYP81A6 and thus renders transgenic rice to be sensitive to bentazon, a herbicide used for rice weed control. We generated transgenic rice plants by this method using a new glyphosate resistant 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene from Pesudomonas putida as the gene of interest, and demonstrated that these transgenic rice plants were highly sensitive to bentazon but tolerant to glyphosate, which is exactly the opposite of conventional rice. Field trial of these transgenic rice plants further confirmed that they can be selectively killed at 100% by one spray of bentazon at a regular dose used for conventional rice weed control. Furthermore, we found that the terminable transgenic rice created in this study shows no difference in growth, development and yield compared to its non-transgenic control. Therefore, this method of creating transgenic rice constitutes a novel strategy of transgene containment, which appears simple, reliable and inexpensive for implementation.

Show MeSH
Related in: MedlinePlus