| The expression of ERD15(Early Responsive to Dehydration)in plants is stimulated by adversity,it plays an important role in plant growth and development in response to various stresses.In this study,the functions of GmERD15 a,GmERD15 b and GmERD15 c were preliminarily explored through bioinformatics analysis,tissue expression analysis and stress resistance identification in yeast system.On this basis,we chose to conduct further research on GmERD15 c,and further explore the function of GmERD15 c through he analysis of gene expression under adverse,transcriptional activation activity analysis and function exploration in transgenic plants.The findings are as follows:1.qRT-PCR was used to successfully detect the expression of GmERD15 in different tissues of soybean.The results showed that GmERD15 a,GmERD15 b and GmERD15 c were expressed in different tissues of soybean,and the expression levels of GmERD15 a and GmERD15 c were the highest in embryo on 20 d,and GmERD15 b was the highest in root tissue.Therefore,GmERD15 a,GmERD15 b and GmERD15 c have no tissue expression specificity.2.The stress resistance of GmERD15 a,GmERD15 b and GmERD15 c was identified in the yeast system,and the results showed that: The growth of yeast cells contained p YES2-GmERD15 a and p YES2-GmERD15 b on drought stress and salt stress medium was worse than that of control yeast,but there was no significant difference on alkali stress medium.The yeast cells transferred into p YES2-GmERD15 c grew better than the control yeast on the medium of drought stress and salt stress.So GmERD15 a and GmERD15 b were sensitive to drought stress and salt stress,while GmERD15 c was tolerant to drought stress and salt stress.3.qRT-PCR was used to successfully detect the expression of GmERD15 in soybean under abiotic stresses.The results showed that the expression level of GmERD15 c was the highest in soybean root tissues under salt stress and salt-alkali stress for 3 h,drought stress for 1h,and ABA treatment for 6 h.The expression of GmERD15 c in soybean leaves was up-regulated to varying degrees under 1/4 h of four kinds of abiotic stress,but decreased after1/2 h and was lower than that in the control group.Therefore,the expression of GmERD15 c gene in soybean roots and leaves is related to stress induction.4.Transcriptional activation of GmERD15 c was verified in yeast.First,the vector of full-length of GmERD15 c was constructed,and it was found that GmERD15 c had transcriptional activation activity by yeast verification.Then,the vectors with different regions of 1-50 aa,1-70 aa,1-83 aa and 1-100 aa were constructed.The results showed that the four different regions had transcriptional activation activity.According to the above experimental results,the carrier of 51-125 aa segment was constructed,and it was found that this segment lost transcriptional activation activity,so 1-50 aa was preliminarily identified as the key transcriptional activation region of GmERD15 c.5.Transgenic Arabidopsis thaliana was obtained by Floral-Dip.11 strains of T1 overexpressed Arabidopsis were obtained by screening and genomic PCR identification,and 10 strains were found to conform to Mendel’s separation law by isolation ratio identification.Three highly expressed lines were screened by real-time fluorescence quantitative method for plate phenotype analysis.It was found that the germination rate of OE strains on the stress medium was higher than that of WT,and the seedlings of OE strain on the stress medium grew more better than WT.This study provided a preliminary basis for phenotypic analysis of transgenic Arabidopsis thaliana with GmERD15 c.6.Positive plants were obtained by genetic transformation of soybean,and 3 T0 generation soybean positive plants were obtained by Bar dipstick test.After seed harvesting,T1 generation soybean lines were planted,and positive plants were detected by genomic PCR.At present,T1 transgenic soybean seeds have been obtained.It provides experimental materials for phenotypic analysis of transgenic soybean with GmERD15 c. |
PDF Full Text Request