| PxbHLH01(KJ466370)and PxbHLH02(KJ466371)are two homologous genes of bHLH transcription factor family of Populus simonii ×P.nigra.In order to reveal the function of two genes of PxbHLH10/02 in response to stress,we studied the promoter activity of PxbHLH01/02 genes,we also analyzed the heterologous expression of PxbHLH01/02 in Arabidopsis thaliana and the overexpression of PxbHLH02 in Populus simonii ×P.nigra.The main findings are as follows:1.The plant transient expression vector of promoter was constructed,and the PxbHLH01/02::GUS recombinant vector was transformed into tobacco plants instantaneously.The results of GUS staining on transgenic tobacco plants showed that the promoter of PxbHLH01/02 had high activity in leaves,and the effects of abiotic stress and hormones on the activity of GUS in tobacco were further studied.The results showed that the promoter had obvious response to NaCl,GA,H2O2,ABA,PEG and 37? treatment.2.The recombinant vectors 35S::PxbHLH01 and 35S::PxbHLH02 were transformed into wild-type Arabidopsis thaliana and upbl mutants to obtain transgenic plants.We studied the phenotype of root length of wild type Arabidopsis thaliana,upbl mutants,PxbHLH01/PxbHLH02 overexpression transgenic Arabidopsis thaliana and PxbHLH01/PxbHLH02 overexpression in upbl mutants.The results showed that both PxbHLHO1 and PxbHLH02 genes could restore the phenotype of upbl mutant root length,which indicated that PxbHLH01/02 and AtUPB1 were homologous genes and functionally similar.We treated PxbHLH01/PxbHLH02 transgenic Arabidopsis thaliana and wild type under stress.The results showed that the germination rate of transgenic Arabidopsis thaliana was earlier than that of wild type for 0.5?4.5 days under 37 ?,NaCl,ABA and drought treatments.The cotyledon greening time of transgenic Arabidopsis thaliana was earlier than that of wild type for 2?3.5days under ABA,NaCl and drought treatments;The root length of PxbHLH01/PxbHLH02 transgenic Arabidopsis thaliana was significantly higher than that of wild type under NaCl and drought treatments,which was 1.56/1.84 times that of wild type and 2.28/2.09 times of wild type.The results indicated that PxbHLH01/02 genes increased the tolerance of Arabidopsis thaliana to stress.Subsequently,we further studied the expression levels of stress related genes AtABIl,AtABI2,AtABI3,AtSODl,AtSOD2,AtMYB2,AtOST1,AtDREB2A and AtNCED3 in transgenic Arabidopsis thaliana and wild-type.The expression of stress related genes in transgenic Arabidopsis thaliana was significantly higher than that in wild type except the exception of AtSOD2 gene,and the highest expression level was 82.84 times of wild type.The results showed that PxbHLH01/02 genes could regulate the expression of stress related genes and increase plant resistance to stress.3.The PxbHLH02 transgenic Populus simonii ×P.nigra and WT were treated with NaCl,mannitol,ABA and 37?.The results showed that under the stress condition,the WT Populus simonii ×P.nigra plants showed curly and yellow leaves,and the leaves of transgenic Populus simonii ×P.nigra showed more green and less damage.The activities of SOD and POD of transgenic Populus simonii ×P.nigra were 1.25 times and 5.54 times higher than those of the control after stress treatment.The H2O2 content,MDA content and relative electrical conductivity of the control group were significantly higher than those of the transgenic plants,and the highest values were 1.24 times,1.51 times and 1.46 times of the transgenic plants.Meanwhile,the transgenic Populus simonii ×P.nigra and the control group were stained with DAB and NBT.The results showed that under drought and 37 ? treatments,the leaves of the control group showed a larger area dyeing,indicating that the overexpression of PxbHLH02 gene increased the resistance of Populus simonii ×P.nigra to 37 ? high temperature,NaCI,drought and ABA stress.The analysis of the expression levels of stress related genes PxABA1,PxABA2,PxABA3,PxSODI,PxSOD2,PxMYB2,PxOST1,PxDREB2A and PxNCED3 in transgenic Populus simonii ×P.nigra and control group showed that the expression of stress-related genes in transgenic Populus simonii×P.nigra was upregulated at different time points,indicating that PxbHLH02 gene could regulate the expression of stress-related genes and increase the resistance of plants under abiotic stress. |
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