Objsective: Glycyrrhizia uralensis, one of the most widely-used traditional Chinese medicines, is mainly cropped in China. However, many cultivars are less in glycyrrhizic acid than Chinese Pharmacopoeia requires. In ...Objsective: Glycyrrhizia uralensis, one of the most widely-used traditional Chinese medicines, is mainly cropped in China. However, many cultivars are less in glycyrrhizic acid than Chinese Pharmacopoeia requires. In this paper, we improved glycyrrhizic acid by regulating β-amyrin synthase gene(GuBAS).Methods: Tobacco root-specific promoter TobRB7 and Gu BAS c DNA were obtained and combined with linearized pCAMBIA1305.1 to construct root-specific plant expression vector which was later transformed into Agrobacterium rhizogenes ACCC10060 by electrotransformation. The cotyledons and hypocotyls of G.uralensis were infected by the recombinant A. rhizogenes ACCC10060 to induce hairy roots. The GA content was quantified by HPLC.Results: The PCR and sequencing results both showed that three transgenic hairy root lines were obtained. The copy number of Gu BAS in these transgenic hairy roots was intended by q RT-PCR to be 3, 7,and 4. GA was detected by HPLC, and the results showed that GA was present in the three transgenic hairy roots, while absent in wild hairy roots.Conclusion: Over-expressing Gu BAS root-specifically in hairy roots of G. uralensis enhanced GA accumulation.展开更多
Two different length fragments, RSF1 and RSF2 which contained the cis-acting sequences of root-specific gene TobRB7, were isolated from tobacco genome. The abilities of these fragments to direct root-specific expressi...Two different length fragments, RSF1 and RSF2 which contained the cis-acting sequences of root-specific gene TobRB7, were isolated from tobacco genome. The abilities of these fragments to direct root-specific expression were studied by fusing them to the β-glucuronidase (GUS) report gene with different directions. After the recombined vectors were transformed into tobacco, the expression pattern was performed by histochemical staining and the quantitative analysis of GUS activity. The data suggested that the cis-acting element of TobRB7 gene direct GUS expression not only as root-specific but also as bidirectional. In our studies, the short fragment, RSF2, performed stronger activity than RSF1 with any direction. The stronger activity of GUS expression was determined by reverse inserting of RSF1 or RSF2 than positive inserting.展开更多
Novel promoters that confer root-specific expression would be useful for engineering resistance against problems of nutrient and water absorption by roots. In this study, the reverse transcriptase polymerase chain rea...Novel promoters that confer root-specific expression would be useful for engineering resistance against problems of nutrient and water absorption by roots. In this study, the reverse transcriptase polymerase chain reaction was used to identify seven genes with root-specific expression in rice. The isolation and characterization of upstream promoter regions of five selected genes rice root-specific promoter （rRSP） 1 to 5 （rRSP1-rRSP5） and A2P （the promoter of OsAct2） revealed that rRSP1, rRSP3, and rRSP5 are particularly important with respect to root-specific activities. Furthermore, rRSP1, rRSP3, and rRSP5 were observed to make different contributions to root activities in various species. These three promoters could be used for root-specific enhancement of target gene（s）.展开更多
基金supported by the National Natural Science Foundation of China (81503181)
文摘Objsective: Glycyrrhizia uralensis, one of the most widely-used traditional Chinese medicines, is mainly cropped in China. However, many cultivars are less in glycyrrhizic acid than Chinese Pharmacopoeia requires. In this paper, we improved glycyrrhizic acid by regulating β-amyrin synthase gene(GuBAS).Methods: Tobacco root-specific promoter TobRB7 and Gu BAS c DNA were obtained and combined with linearized pCAMBIA1305.1 to construct root-specific plant expression vector which was later transformed into Agrobacterium rhizogenes ACCC10060 by electrotransformation. The cotyledons and hypocotyls of G.uralensis were infected by the recombinant A. rhizogenes ACCC10060 to induce hairy roots. The GA content was quantified by HPLC.Results: The PCR and sequencing results both showed that three transgenic hairy root lines were obtained. The copy number of Gu BAS in these transgenic hairy roots was intended by q RT-PCR to be 3, 7,and 4. GA was detected by HPLC, and the results showed that GA was present in the three transgenic hairy roots, while absent in wild hairy roots.Conclusion: Over-expressing Gu BAS root-specifically in hairy roots of G. uralensis enhanced GA accumulation.
文摘Two different length fragments, RSF1 and RSF2 which contained the cis-acting sequences of root-specific gene TobRB7, were isolated from tobacco genome. The abilities of these fragments to direct root-specific expression were studied by fusing them to the β-glucuronidase (GUS) report gene with different directions. After the recombined vectors were transformed into tobacco, the expression pattern was performed by histochemical staining and the quantitative analysis of GUS activity. The data suggested that the cis-acting element of TobRB7 gene direct GUS expression not only as root-specific but also as bidirectional. In our studies, the short fragment, RSF2, performed stronger activity than RSF1 with any direction. The stronger activity of GUS expression was determined by reverse inserting of RSF1 or RSF2 than positive inserting.
基金supported by the National Natural Science Foundation of China (31271694)the National Transgenic Plant Program of China (2011ZX08001-003)
文摘Novel promoters that confer root-specific expression would be useful for engineering resistance against problems of nutrient and water absorption by roots. In this study, the reverse transcriptase polymerase chain reaction was used to identify seven genes with root-specific expression in rice. The isolation and characterization of upstream promoter regions of five selected genes rice root-specific promoter （rRSP） 1 to 5 （rRSP1-rRSP5） and A2P （the promoter of OsAct2） revealed that rRSP1, rRSP3, and rRSP5 are particularly important with respect to root-specific activities. Furthermore, rRSP1, rRSP3, and rRSP5 were observed to make different contributions to root activities in various species. These three promoters could be used for root-specific enhancement of target gene（s）.
