Screening And Functional Analysis Of Drought Response Genes In Picea Wilsonii

Picea wilsonii,also known as Weiss spruce or North China spruce,belongs to the genus Picea of Pinaceae.It is a unique evergreen coniferous tree species in China,and is also an important landscape tree species and timber forest species.It has a very high ecological value.With the deterioration of the global environment,drought has become one of the main environmental factors affecting ecological balance and agricultural and forestry production worldwide.The living environment of Picea wilsonii is threatened,and its distribution is decreasing year by year.Therefore,it is essential to improve the drought tolerance of Picea wilsonii by means of molecular biology and other means.In this study,the target genes responding to drought stress were screened from the transcriptome data of Caucasus sinensis using Protein BLAST function of NCBI website and RT-q PCR.The drought tolerance of the target gene was studied and validated by stress and hormone treatment,tissue specificity analysis,chromosome walking,yeast two-hybrid,subcellular localization and gene transformation.The main research results are as follows:1.In this study,five drought-tolerant genes were screened from the transcriptome of Picea wilsonii,namely PwPEBP,PwMBF1c,PwRhomboid,PwWD40 and PwREF,and it was analyzed for tissue specificity and expression levels under stress and hormone treatment.The results showed that the five genes all had the highest expression in the mature needles of the Picea wilsonii.In the stress and hormone treatment,all the five genes responded strongly to drought stress.Under the induction of ABA,the expression of genes increased or decreased significantly.In addition to responding to drought stress and ABA treatment,PwRhomboid gene expression increased significantly under low temperature and SA treatment,and PwMBF1c gene expression also increased significantly under high temperature,MeJA and SA treatment,in which the expression of PwMBF1c gene increased significantly to 104 times of the control under high temperature induction.2.Subcellular localization of three target proteins was performed by injecting Agrobacterium tumefaciens into tobacco leaves.The results showed that PwPEBP and PwMBFlc were mainly localized in the nucleus and cytoplasm,while PwRhomboid was localized in the cell membrane.3.The 903 bp promoter of PwPEBP was cloned by chromosome walking method.The results of promoter activity analysis showed that PwPEBP promoter had strong response to drought induction,and PwMBF1c terminal had self-activation activity in yeast two-hybrid test.4.In transgenic potatoes,the seedlings of PwPEBP,PwMBF1c and PwRhomboid treated with 15%and 20%PEG6000 were higher in height and growth than those of wild type,which indicated that these three genes could improve drought tolerance of plants.5.In the seedling test of two lines of Arabidopsis thaliana with PwMBFlc gene,under different concentration of mannitol stress,the root length of overexpressed lines was longer than that of wild type and PCM 1205 lines,but the seed germination rate was slightly lower than that of wild type lines.After 7 days of seedling drought treatment,the chlorophyll content of transgenic lines was significantly lower than that of wild type and PCM 1205 lines,wild type and PCM 1205 lines.The content of PwMBF1c was also significantly higher than that of the transgenic lines.After rewatering 16 days after drought treatment,the survival rate of the transgenic lines reached more than 86%,which was significantly higher than that of the wild type lines.This further indicated that PwMBF1c gene could improve the drought tolerance of plants.