Function Analysis Of Populus Euphratica Brassinosteroids Biosynthase Gene CPD By RNA Interference

Brassinosteroids (BRs) are a new type of plant hormones in a wide plant kingdom, which play a very important role in multiple aspects of development, such as cell expansion and division, senescence, male fertility and response to stress. So far, researches concerning the biosynthesis and signal transduction of BRs mainly focus on the herb plants instead of woody plants. CPD (constitutive photomorphogenesis and dwarf) gene encodes a key enzyme in BR biosynthesis. In the present paper, Populus euphratica CPD gene (PeCPD) was used to construct RNA interference (RNAi) vector pAR2, which was used to transform Arabidopsis thaliana wide type (WT). Three homozygous RNAi transgenic plants (named R-9, R-12and R-13) were gained. To understand the function of PeCPD, we also analysed the phenotypes, physiology and the expressions of AtCPD and other BR-related genes. The main results are as follows.1. All the RNAi transgenic plants, especially R-12, showed growth lag to different extent:the vegetative period of R-12laged5～7days and plant height in the reproductive growth stage was decreased by33.5%on average, compared with WT.2. The elongation rate of root in RNAi plants of T2generation was obviously reduced in R-9and R-12, whose relative root lengths were respectively68.82%and63.67%of WT. In the presence of0.05nM BL, their relative root lengths reached to92.75%and96.82%of WT.3. In the dark, the elongation rate of hypocotyl of R-12line was decreased, which was11.3%shorter than that of WT.4. The resistance of R-12line to salt-stress was distinctively diminished relative to WT: in the presence of different concentration salt, seed germination time lagged1～2days and the maximum germination rate was10%～20%lower than WT; What’s more, R-12seedlings grown on solid medium showed an early curly and brown leaves compared with WT, and the relative root lengths were decreased by30%～35%.5. Real-time PCR detecting the expression level of AtCPD and other BR-related genes in the RNAi transgenic lines showed that expression level of AtCPD was71%of WT, with no apparent differences in R-9and R-13. The expression level of DWF4(down-regulated by endogenous BR) was similar to that of WT, while the expression level of BR6OX2(down-regulated by endogenous BR) was between that of cpd and WT. By contrast, the expression levels of SAU-AC1and BAS1(both upregulated by exogenous BR) were both between that of cpd and WT.Results above suggested that PeCPD gene may be the same function as AtCPD. All the changes in phenotypes may be related to the reduced endogenous BR level induced by RNAi, suggesting that PeCPD plays a critical role in BR biosynthesis in Populus euphratica.