Identification Of Glycine Max Steroid Dehydrogenase Gene Family And Functional Analysis Of GmDET2a And GmDET2b

Brassinosteroid(BRs)is a kind of classic phytohormone,which play important roles in many aspects of plant growth anddevelopment.Of note,sterol dehydrogenase is a key enzyme that is involved in the biosynthesis of BR.Arabidopsis thaliana DET2(At DET2)is a well-studied sterol dehydrogenase,and loss of function mutations in At DET2 results in dwarfing and fertility reduction in Arabidopsis.Soybean(Glycine max)is an important grain and oil crop world wide.It has been shown that BRs regulate growth and stress tolerance of soybean,but the underlying molecular mechanisms ofBRbiosynthesis in soybean is not fully understood.In this study,we firstly identified soybean sterol dehydrogenase gene family,reconstructed the phylogenetic tree,and analyzed the selection pressure of sterol dehydrogenase in Planta.Then we explored the responses of soybean DET2 a and DET2 b to nutrient stresses and exogenous BR.Last genetic complementation experiments verified that Gm DET2 a and Gm DET2 b had similar functions to Arabidopsis DET2 gene.The main results are as follows:(1)Soybean genome contains 8 sterol dehydrogenase genes which are named as Gm DET2a-2h,among them,Gm DET2 a and Gm DET2 b genes are located on chromosomes 7 and 11,which both encode proteins with 263 amino acid residues,respectively.Evolutionary analysis showed that GmDET2 a and GmDET2 b are highly homology with PsDET2 and AtDET2.We also reconstructed the phylogenetic tree of 105 sterol dehydrogenases from algae,fern and higher plants and found that the sterol dehydrogenase family was divided into five subgroups.Moreover,sterol dehydrogenase family aresubjected to purifying selection during evolutionary process.(2)Real time quantitative RT-PCR showed that Gm DET2 a and Gm DET2 b ubiquitously expressed in all tested soybean organs in seedling stage.At seedling stage,the expression levels of Gm DET2 a in primary root and apical bud were significantly higher than that of other parts.The expression level of Gm DET2 b in primary root,apical bud and lateral roots were also significantly higher than that in other parts.(3)In contrast to control(noEBLapplication),application ofEBLdown-regulated the transcript levels of Gm DET2 a in the apical bud,euphylla,primary root and lateral roots of YC03-3 by 38%,55%,70% and 67%,respectively.O n the other hand,the abundance of Gm DET2 b in primary root and lateral roots were all significantly decreased by the application ofEBLin YC03-3 by 61%.These results indicate that the expression of Gm DET2 aand Gm DET2 b is negatively regulated by exogenous BR.(4)Expression patterns of Gm DET2 a and Gm DET2 b under different nutrient conditions in seedling stage were also studied.Results showed that in contrast to high sulfur conditions,the expression of Gm DET2 a was significantly decreased by 63% in sulfur deficiency,and the transcripts of Gm DET2 b in leaves and roots were significantly lower,which were reduced by 55% and 86%,respectively.Moreover,low phosphate induced expression of Gm DET2 b in leaves and roots by 70% and 89%,respectively.However,the transcript levels of Gm DET2 a and Gm DET2 b did not respond to nitrogen(N)and potassium(K)deficiency.(5)Subcellular localization with fused GFPindicated that GmDET2 a and GmDET2 b appear to be localized in endoplasmic reticulum.(6)Over-expressing Gm DET2 a and Gm DET2 b in mutant det2-1mutant,resulted in higher plant height,which is similar to Col-0.This difference was obviously found in Gm DET2aOX-23 or Gm DET2bOX-16 line.More importantly,the transcript levels of CPD,DWF4,BR6ox1 and BR6ox2 genes,which are involved in BRs biosynthesis,were down-regulated in different extent compared with det2-1,and their expression levels were similar to those in wild type Col-0.(7)In darkness,compared to det2-1,the length of hypocotyls of Gm DET2aOX-20 and Gm DET2aOX-23,Gm DET2bOX-16 and Gm DET2bOX-23 were increased by 110%,87%,146% and 121%,respectively,and no difference were found between Col-0 and Gm DET2aOX-20 or Gm DET2aOX-23 or Gm DET2bOX-16 or Gm DET2bOX-23.In summary,we identified 8 soybean sterol dehydrogenase,and revealed that plant sterol dehydrogenase are grouped into 5 subgroups and were subjected to purifying selection during evolution.The functions of DET2 a and DET2 b are similar to the counterpart of Arabidopsis DET2.These results paved a way to the further studies on the molecular mechanisms of soybeanBRbiosynthesis and the function of Gm DET2.