Establishment Of Agrobacterium Mediated Transformation System For Nepeta Multifida And The Introduction Of Cjhppd Gene

HPPD became a new target for herbicide in 1990 s.It can catalyzes the formation of plastoquinone and vitamin E in the plants.Cjhppd was resistant to herbicide DTP.It was cloned from cell lines in high yield of Coptics japonica and encoded Coptis japonica HPPD gene.The study was aimed to integrate the Cjhppd gene into the genome of Nepeta multifida and obtain the transgenic plants.The study explored the effects of Cjhppd gene on resistance traits of transgenic plants.The Agrobacterium-mediated transformation system for Nepeta multifida was established in this study.The Agrobacterium tumefacien strain used in this study was the LBA4404 which carrying with the pBI121-gfp plasmid.The segment of stem with axils was used as explants.The factors effecting on transgenic efficiency,such as pre-incubation time,bacterial concentration,infection time,co-culture time were studied.Each factor was set up with four levels.L16(45)orthogonal experiment was conducted three times independently.The transgenic buds and Nepeta multifida were confirmed by PCR,RT-PCR,GFP fluorescence detection.In this section,the Agrobacterium tumefacien strain would infect the stem with axils.The Agrobacterium tumefacien strain used in this study was the LBA4404 which carrying with the PGWB2-Cjhppd plasmid.The aim was that the Cjhppd gene could integrate in the genome of the transgenic plants.The Cjhppd transgenic plants were confirmed by PCR,RT-PCR assays.Then the transgenic plants treated by different concerntration NaCl and DTP were used to study the ability of resistance to DTP and NaCl of transgenic plants.The main results were summarized as follows:1.The results showed that the gfp gene was successfully integrated in the genome of the transgenic plants.The expression of gfp gene was highest in the root,then stem and leaf,flowers of transformed plants.The optimum condition for transformation was pre-culture for5 days,Agrobacterium OD600 concerntration at 0.7,infection for 15 minutes,co-culture for 3days.The transgenic efficiency was 20%,and the average number of transgenic buds was 5 per explant.The rooting efficiency was 60%.The establishment of Nepeta multifidatransformation system lays the groundwork for the improvement of the genetic traits via plant genetic engineering.2.Moved the transgenic Cjhppd buds into root medium to induce rooting,after selecting for 30 days.After one month,the transgenic plants were obtained.The transgenic rate was22%.The rooting rate was 66%.3.The PCR and RT-PCR detection of Cjhppd showed that Cjhppd gene was successfully integrated in the genome of the transgenic plants.The expression of Cjhppd gene was highest in the root,then stem and leaf.The transgenic plants were moved to soil when they growed to5-6 centimeters.The transplanting rate was 60%.4.The result of the herbicide resistance in Cjhppd transgenic plants indicated that wild plants leaves bleaching was more severe than that of transgenic type at the same concerntration of DTP.The results showed the transgenic plants had some tolerance to DTP.5.The result of the NaCl resistance in Cjhppd transgenic plants showed the transgenic plants could grow healthy at 150 mmol/L NaCl concentration,but at 200 mmol/Lwas wilting.The wild type plants at 50 mmol/L appeared yellow leaves wilting phenomenon.The variation of Pro,POD,MDA,CAT rised first and then falled in the wild and transgenic plants.But the speed of transgenic plants was significantly lower than that of wild type.The chlorophy ll carotenoid content of wild Nepeta multifida was significantly lower than before,while rised first and then falled in the transgenic plantes and significantly higher at the same concerntration of NaCl.The NaCl had a great influence on carotenoid content.The results showed that the transgenic plants had some tolerance to salt stress.The salt tolerance concerntration was 150 mmol/L.The carotenoid might be the factor to improve the salt tolerance of transgenic plants.