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Identification And Functional Analysis Of Transgenic Tobacco Of Suaeda Salsa Dehydrin Gene

Posted on:2017-02-26Degree:MasterType:Thesis
Country:ChinaCandidate:M SunFull Text:PDF
GTID:2180330485972347Subject:Cell biology
Abstract/Summary:PDF Full Text Request
In this study, transgenic tobacco of Suaeda salsa DHN and tobacco of the wild-type are choosed as research materials, and transgenic plants are detected at the level of DNA molecular, protein expression and physiological, in order to obtain transgenic tobacco of Suaeda salsa DHN which provides reliable materials for the further study on the function and mechanism of the gene.PCR and Southern blot are included in the identification of transgenic tobacco at the molecular level. The DNA of transgenic tobacco, of wild-type tobacco, and the plasmid of PBI121-DNH-Agrobacterium are extracted. The plasmid of PBI121-DNH-Agrobacterium is used as positive control and the DNA of wild-type tobacco is used as negative control. PCR is processed under the role of specific primers. Six positive DHN-transgenic tobaccos are detected after PCR. But as PCR detection may produce false positive results, Southern blot is carried out in the future. The plasmid of PBI121-DNH-Agrobacterium is marked as a probe, which is used to hybridize with positive control, negative control and the DNA of transgenic tobacco. The CSPD method is used to develop hybridization signals. Chromogenic results showed that the six positive transgenic tobacco of PCR detection have obvious hybridization signal, which indicates that the DHN gene is successfully transfered into tobacco genome.Prokaryotic expression. PAGE and Western blot are carried out in the identification of transgenic tobacco on protein levels. The protein of transgenic tobacco which are detected by PCR and Southern blot, of wild-type tobacco and of pET28b-SsDHN-E.coliBL21 are extracted. The protein of pET28b-SsDHN-E.coliBL21 is used as positive control and the protein of wild-type tobacco is used as negative control. Different molecular weight protein is segregated in the PAGE. The DHN protein band is screened according to the theoretical molecular weight (27KD) of DHN protein, and the electrophoresis results show that six tobacco have bands at 27KD. which indicates that the DHN protein are expressed. The bands at 27KD are used to hybridize with anti-dehydin (primary antibodies). And then "DHN-primary antibodies" hybridize with alkaline phosphatase-labeled secondary antibody. The BCIP/NBT method is used to develop hybridization signals. Test results show that the protein bands at 27KD band all have hybridization signals, indicating that the six transgenic tobacco expressed DHN protein.Relative conductivity, MDA content, SOD activity and free proline content were included in the analysis of physiological function of transgenic tobacco. With the increase of salt concentration, the relative conductivity and MDA content of both transgenic tobacco and wild-type tobacco are increasing, but the relative conductivity and MDA content of transgenic tobacco is lower than the wild-type tobacco at the same concentration; the SOD activity of transgenic tobacco is increasing and the SOD activity of wild-type tobacco increases first and then decreases, only the SOD activity of transgenic tobacco is higher than the wild-type tobacco at the same concentration; the free proline content of both transgenic tobacco and wild-type tobacco increases first and then decreases, but the free proline content of transgenic tobacco is higher than the wild-type tobacco at the same concentration. The results show that the salt tolerance of transgenic tobacco is better than the wild-type tobacco, which indicates that the transgenic tobacco of Suaeda salsa DHN has the function of salt tolerance.
Keywords/Search Tags:Dehydrin, Tobacco, Southern blot, Western blot, Physiological function
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