Functional Research Of NtabSPL6-2 And NtabSPL6-3 Genes In Nicotiana Tabacum

The main content of this work is identification of the function of NtabSPL6-2 and NtabSPL6-3 genes.In preparation of transgenic Arabidopsis plants,floral dip method mediated by Agrobacterium was adopted,NtabSPL6-2 and NtabSPL6-3 were integrated into the genome of Arabidopsis,and transgenic plants were acquired by selection with kanamycin.By observation of the phenotypic changes in the transgenic plants and detection of the expression levels of the downstream genes of SPL genes in the aging pathway of flowering in Arabidopsis thaliana,the biological functions of NtabSPL6-2 and NtabSPL6-3 were analyzed preliminarily.Compared to the wild-type Arabidopsis plants,the number of the rosette leaves in NtabSPL6-2 and NtabSPL6-3 transgenic plants have no significantly difference,but the cauline leaves were converted into opposite leaves from single blade.In NtabSPL6-2transgenic plants,the sixth,the seventh,and the eighth rosette leaves generated irregularly serrate margin.Moreover,the leaves of NtabSPL6-2 transgenic plants were larger and had a significantly higher fresh,accompanied with thicker and longer roots.Furthermore,subcellular localization using GFP gene confirmed that the proteins encoded by NtabSPL6-2and NtabSPL6-3 existed in the nucleus.In addition,the structures of NtabSPL6-2 and NtabSPL6-3 were predicted by using online protein modeling software.In order to determine the roles of NtabSPL6-2 in plant resistance against pathogens,NtabSPL6-2 transgenic Arabidopsis plants were infected with Pseudomonas syringae.the infected leaves of NtabSPL6-2 transgenic plants had smaller injured area,less wilting degree and a lower level of bacterial reproduction than that of the wild-type plants.The data of qRT-PCR showed that the expression level of the resistance-related gene PR1 in the infected leaves of NtabSPL6-2 transgenic plants was also higher,indicating NtabSPL6-2 could produce a certain resistance response to P.syringae at the local infection sites.Similarly,it was also confirmed that NtabSPL6-2 could enhance the systemic acquired resistance response after infection with P.syringae.To illuminate the resistance response of NtabSPL6-2 transgenic plants and the wild-type Arabidopsis plants to local infection of Botrytis cinerea,the area of necrotic spots appearing on the leaves after infection was quantitatively analyzed.Compared to the infected leaves of the wild-type plants,the infected leaves of the NtabSPL6-2 transgenic plants were less susceptible to B.cinerea,and displayed a weaker wilting degree and a smaller lesion area.Moreover,the expression levels of the resistance-related genes PR1 and PR5 in the infected leaves of NtabSPL6-2 transgenic plants were higher than the wild-type plants.It was also found that less amounts of ROS?H₂O₂ and O₂^-?and MDA were accumulated in the infected sites of the leaves of transgenic plants.These results certified that the NtabSPL6-2 transgenic plants were less oxidatively damaged than the wild-type plants after local infestation with B.cinerea.The results also manifested,comparison with the wild-type plants,that NtabSPL6-2transgenic plants showed stronger defensive response to B.cinerea.At the same time,this work also analyzed the response characteristics of NtabSPL6-2transgenic Arabidopsis plants to salt stress conditions,and observed the germination rate of NtabSPL6-2 transgenic seeds and wild-type seeds.On normal medium,the germination rate of NtabSPL6-2 transgenic seeds was higher than that of wild-type seeds.When NaCl concentration was 50 mmol/L and 100 mmol/L,the germination rate of NtabSPL6-2transgenic seeds was higher than that of wild-type seeds.when the NaCl concentration was 50mmol/L and 100 mmol/L,the electrolyte leakage of the leaves of NtabSPL6-2 transgenic seedlings was lower than that of wild-type seedlings,indicating that the damage of NaCl to NtabSPL6-2 transgenic plants was less severe in comparison with the wild-type plants.The H₂O₂ content in the leaves of the seedlings was also measured in the present work.When the NaCl concentration was 50 mmol/L or 100 mmol/L,the H₂O₂ content of the leaves of NtabSPL6-2 transgenic plants was lower than that of the wild-type plants.These results demonstrated that NtabSPL6-2 could enhance the tolerance of Arabidopsis to salt stress.