Manipulation Of Cellular Cam Levels Of Transgenic Tobacco Proline Metabolism

Transgenic tobacco plants that express CaM genes have been developed by our lab to study the relationship between CaM and proline metabolism. This research includes three parts:1. Screening of transgenic tobacco lines expressing nuclear targeted CaMThe CaM activity of 26 transgenic tobacco lines expressing nuclear targeted CaM were measured by phosphodiesterase, then we selected four good transgenic tobacco lines(T2.1, T2.25, T2.19 and T2.34) whose CaM activity were increased significantly (about 30-42% ) in seedlings after Tc induction.2. The T2.1, T2.25 transgenic tobacco and T1.1 transgenic tobacco expressing cytosolic targeted CaM were used as experimental materials, to study the relationship between CaM and proline metabolism in tobacco seeding. Tc-inducible transgenic tobacco expressing nuclear targeted CaM(T2.1, T2.25) and cytosolic targeted CaM(T1.1) seedlings showed the increased content of proline than those without Tc-induction and the control(T2.3).The more CaM activity of transgenic tobacco was increased, the more proline will be accumulated in these seedings. This is because glutamate pathway was stimulated by Tc-inducible transgenic tobacco expressing cytsolic CaM or nuclear CaM. This effect can be decreased obviously after the CaM inhibitor CPZ treatment.3. At last, the T2.1, T2.25 and T1 .1 transgenic tobacco(90 days) leaf disks were used to study the effect of Tc-inducible transgenic tobacco expressing cytsolic targeted CaM or nuclear targeted CaM on proline metabolism under osmotic stress.The results demonstrated that the proline content in leaf disks increased about 5 times after the treatment of transgentic tobacco with 5% PEG6000 for 96 hours. Interestingly, the proline content of transgenic tobacco leaf disks with Tc induction were increased significantly under osmotic stress, which are higher than those tobacco leaves without Tc induction. The mechanism of CaM regulation on proline metabolism of transgenic tobacco under osmotic stress is different to which under normal growth condition. Both glutamate and ornithine pathways of proline biosynthesis were stimulated by CaM under osmotic stress. It seems that the Orn pathway is more regulated by CaM than Glu pathway in proline accumulation of transgenic tobacco under osmotic stress.Proline content can be decrease obviously by CPZ treatment.According to above, CaM might play an important role in proline metabolism of transgenic tobacco.