Studies On The Evolution And Function Of Brassinosteroid Synthetic Genes In Selaginella Jiangnanensis

Brassinosteroids(BRs),recognized as the sixth type plant hormones,are widely found in angiosperms,gymnosperms,ferns,bryophytes and green algae.So far,more than 70 BRs have been discovered.BRs play a key regulatory role in the important processes of plant seed dormancy and germination,organ differentiation,vascular tissue development,flowering and senescence,and tropism.In recent years,BR biosynthesis,metabolism and signaling pathways in seed plants,such as Arabidopsis and rice,have been well studied,but their role in seedless plants is not clear.In this thesis,the biosynthetic genes from Selaginella moellendorffii were studied.Phylogenetic analysis was first applied to study these genes,followed by function study in Arabidopsis thaliana wild type Col-0 and det2,cpd,rot3,dwf4 and other BR synthetic mutants through Agrobacterium-mediated gene transformation technology.These heterogeneously expressed genes were then studied by phenotypical and physiological characterization,laser confocal scanning microscopy(LSCM)and Semi-qRT-PCR techniques.The results are as follows:1.The DET2 gene is highly conserved,and the SmDET2 gene of S.moellendorffii can restore the phenotypes of det2.In dark conditions,the det2 mutants exhibited short and thick hypocotyls with open cotyledons.Under the light,the leaf petioles of the det2 seedlings were short while the leaves were rounder and stacked tighter.Furthermore,the mature plant height is also shorter while the length of the pods and the number of seeds in each single pod were significantly lower than that of the wild type.When transferred the SmDET2 gene into this mutant,the phenotypes of the transgenic plants resembled the wild type,which includes all above aspects.In addition,the SmDET2/det2 plants and the wild-type Col-0 had similar sensitive to the BR synthesis inhibitor BRZ.These results indicate that SmDET2 has the similar function as that of AtDET2 and may be involved in the synthesis of BR in S.moellendorffii.2.There are differences in the functions of the CYP90 genes from Selaginella.The expression level of CYP90 genes in the leaf and spore leaf of S.moellendorfii was analyzed by semi-quantitative RT-PCR.It was found that Sm89026 and Sm157387 were highly expressed in both organs,while Sm233379 was only expressed in the leaves,indicating that CYP90 homologous genes may have different functions in S.moellendorfii.3.Ecotopic overexpression of Selaginella CYP90 affects the fertility of A.thaliana.The overexpression of CYP90 homologous genes of S.moellendorfii in wild-type Col-0 revealed that Sm233379 increased the hypocotyl of the seedlings and the height of the mature plants,but shortened the length of the stamen filaments.Most pollen were deformed and the number of pollen were decreased,which eventually resulted in a decrease of seed setting in these transgenic plants.4.SmCPD(SmCYP90A1)has a conserved function that restores the phenotypes of Arabidopsis cpd.Under dark,the cotyledons of the cpd mutant were open with no apical hooks.In the light,the petioles of the plants were extremely short while the leaves were dark green.When SmCPD gene was transferred into this mutant,the transgenic plants resembled Col-0,which had apical hooks and closed cotyledons in the dark.The phenotypes of the SmCPD-OXplants under light conditions are also similar to that of Col-0.Semi-quantitative RT-PCR results showed that SmCPD enhanced the expression of BR metabolic genes but reduced BR biosynthetic genes in A.thaliana,especially the BAS1 and DWF4,respectively.5.The CYP90 gene family is derived during plant evolution.Phylogenetic analysis of CYP90 family genes from the species with completely sequenced genomes found that there was no CYP90C1(ROTS)in the monocots.Importantly,no SmCYP90 could rescue the phenotypes of rot3 mutants.These results suggest that the CYP90C1 gene may be unique in dicots.6.In higher plants,BR function requires its binding to the receptor,but no receptor is found in Selaginella.Therefore,it can be inferred that BR may play a role through a yet unknown receptor,or only as a physiologically active substance to regulate plant growth and development,rather than to exert a hormonal function.Based on the above results,it can be concluded that there is a BR biosynthesis pathway in S.moellendorfii,and DET2 and CPD are involved in the biosynthesis of BR,but no homologs of CYP85A1/A2,DWF4 and ROR3 are found,so BR biosynthesis and signaling pathways in S.moellendorfii may differ from that of angiosperms.However,the enzyme activity of these BR biosynthetic genes and the characteristic and biological activity of the products of these enzymes in S.moellendorfii need further analysis.The CYP90 family involved in BR biosynthesis is derived during the plant evolution,where CYP90C1 is emerged later in plant evolution,and is therefore unique to dicots.