Transferring foreign DNA into plant cells by biolistic and Agrobacterium _mediated methods may result in random integration of different copy numbers of the transgene, and different proportions of intact vs. rearra...Transferring foreign DNA into plant cells by biolistic and Agrobacterium _mediated methods may result in random integration of different copy numbers of the transgene, and different proportions of intact vs. rearranged copies of the transgene. This may, in turn, affect transgene expression levels. To test the above hypothesis, we first introduced the same plasmid, pAc1PG_CAM, into rice (Oryza sativa L.) calli separately by the biolistic method and by the Agrobacterium _mediated method. To show whether different plasmids may affect the results, we also introduced pTOK233 by the Agrobacterium _mediated method and pJPM44 by the biolistic method. Transgene expression of R0 plants was monitored by histochemical analysis of GUS activity. Transgene copy number was determined by Southern blot analysis after digesting genomic DNA with an enzyme that has a unique cutting site within the input plasmid. The total genomic DNA was also digested by a two_cut enzyme (the cuts are located at two sides of a given transgene expression cassette), followed by Southern blotting analysis, for determining the number of intact transgene expression cassettes. Our data showed that Agrobacterium _mediated transformation resulted in lower transgene copy number (average between 2.1 and 2.3) in transgenic rice plants, compared with those plants obtained by the biolistic method (average between 4.2 and 5.6). The frequency of DNA rearrangement in expression cassettes is lower in transgenic rice plants obtained by the Agrobacterium _ mediated method than those obtained by the biolistic method. The average rearrangement frequency is 0.07 to 0.106 for the Agrobacterium _mediated method, and 0.57 to 0.66 for the biolistic method. Our results suggest that it is better to compare the number of intact expression cassettes instead of the total copy number of the transgene in demonstrating their influence on the level of transgene expression. This is the first report on the frequency of expression cassette rearrangement in transgenic plants transformed with the same plasmid by two different transformation methods.展开更多
In order to improve the frequency of indica rice transformation by biolistic bombardment, suitable culture conditions for embryonic calli,an optimal selection scheme for resistant calli and seedlings, and optimum bomb...In order to improve the frequency of indica rice transformation by biolistic bombardment, suitable culture conditions for embryonic calli,an optimal selection scheme for resistant calli and seedlings, and optimum bombardment parameters a investigated by using 14 commercially important indica rice cultivars. The main results show that the CC medium with 36g/L mannitol is a scheme subculture medium in which the browning of indica rice calli can be mitigated significantly; The concentration of 30～40mg/L Hyg or 150～200mg/L G418 or 10～20 mg/L Basta is suitable for selection of resistant calli; The transformation parameters of 100μg gold powder absorbing 0.2μg DNA per shot and 900 psi helium pressure and 6 cm bombardment distance and bombarded twice for each plate give the best result; Keeping the target calli on osmotic medium containing 60g/L mannitol from 12 ～24h before bombardment to 24～48h after it can increase the efficiencies of transformation . Furthermore, some transgenic indica rice plants are obtained using this optimized transformation system.展开更多
This paper considers the compound Poisson risk model perturbed by Brownian motion with variable premium and dependence between claims amounts and inter-claim times via Spearman copula. It is assumed that the insurance...This paper considers the compound Poisson risk model perturbed by Brownian motion with variable premium and dependence between claims amounts and inter-claim times via Spearman copula. It is assumed that the insurance company’s portfolio is governed by two classes of policyholders. On the one hand, the first class where the amount of claims is high, and on the other hand, the second class where the amount of claims is low, this difference in claim amounts has significant implications for the insurance company’s pricing and risk management strategies. When policyholders are in the first class, they pay an insurance premium of a constant amount c<sub>1</sub> and when they are in the second class, the premium paid is a constant amount c<sub>2</sub> such that c<sub>1 </sub>> c<sub>2</sub>. The nature of claims (low or high) is measured via random thresholds . The study in this work will focus on the determination of the integro-differential equations satisfied by Gerber-Shiu functions and their Laplace transforms in the risk model perturbed by Brownian motion with variable premium and dependence between claims amounts and inter-claim times via Spearman copula. .展开更多
文摘Transferring foreign DNA into plant cells by biolistic and Agrobacterium _mediated methods may result in random integration of different copy numbers of the transgene, and different proportions of intact vs. rearranged copies of the transgene. This may, in turn, affect transgene expression levels. To test the above hypothesis, we first introduced the same plasmid, pAc1PG_CAM, into rice (Oryza sativa L.) calli separately by the biolistic method and by the Agrobacterium _mediated method. To show whether different plasmids may affect the results, we also introduced pTOK233 by the Agrobacterium _mediated method and pJPM44 by the biolistic method. Transgene expression of R0 plants was monitored by histochemical analysis of GUS activity. Transgene copy number was determined by Southern blot analysis after digesting genomic DNA with an enzyme that has a unique cutting site within the input plasmid. The total genomic DNA was also digested by a two_cut enzyme (the cuts are located at two sides of a given transgene expression cassette), followed by Southern blotting analysis, for determining the number of intact transgene expression cassettes. Our data showed that Agrobacterium _mediated transformation resulted in lower transgene copy number (average between 2.1 and 2.3) in transgenic rice plants, compared with those plants obtained by the biolistic method (average between 4.2 and 5.6). The frequency of DNA rearrangement in expression cassettes is lower in transgenic rice plants obtained by the Agrobacterium _ mediated method than those obtained by the biolistic method. The average rearrangement frequency is 0.07 to 0.106 for the Agrobacterium _mediated method, and 0.57 to 0.66 for the biolistic method. Our results suggest that it is better to compare the number of intact expression cassettes instead of the total copy number of the transgene in demonstrating their influence on the level of transgene expression. This is the first report on the frequency of expression cassette rearrangement in transgenic plants transformed with the same plasmid by two different transformation methods.
基金the Chinese 863 High-Technology Program(NO.863-101-01-01-01)and Chinese Post-Doctoral Science Foundation(2001-14)
文摘In order to improve the frequency of indica rice transformation by biolistic bombardment, suitable culture conditions for embryonic calli,an optimal selection scheme for resistant calli and seedlings, and optimum bombardment parameters a investigated by using 14 commercially important indica rice cultivars. The main results show that the CC medium with 36g/L mannitol is a scheme subculture medium in which the browning of indica rice calli can be mitigated significantly; The concentration of 30～40mg/L Hyg or 150～200mg/L G418 or 10～20 mg/L Basta is suitable for selection of resistant calli; The transformation parameters of 100μg gold powder absorbing 0.2μg DNA per shot and 900 psi helium pressure and 6 cm bombardment distance and bombarded twice for each plate give the best result; Keeping the target calli on osmotic medium containing 60g/L mannitol from 12 ～24h before bombardment to 24～48h after it can increase the efficiencies of transformation . Furthermore, some transgenic indica rice plants are obtained using this optimized transformation system.
文摘This paper considers the compound Poisson risk model perturbed by Brownian motion with variable premium and dependence between claims amounts and inter-claim times via Spearman copula. It is assumed that the insurance company’s portfolio is governed by two classes of policyholders. On the one hand, the first class where the amount of claims is high, and on the other hand, the second class where the amount of claims is low, this difference in claim amounts has significant implications for the insurance company’s pricing and risk management strategies. When policyholders are in the first class, they pay an insurance premium of a constant amount c<sub>1</sub> and when they are in the second class, the premium paid is a constant amount c<sub>2</sub> such that c<sub>1 </sub>> c<sub>2</sub>. The nature of claims (low or high) is measured via random thresholds . The study in this work will focus on the determination of the integro-differential equations satisfied by Gerber-Shiu functions and their Laplace transforms in the risk model perturbed by Brownian motion with variable premium and dependence between claims amounts and inter-claim times via Spearman copula. .
