Cloning And Genetic Transformation Of A Potato Late Blight Resistance-related Transcription Factor StR2R3

There are always adverse stresses in different situations during the whole growth cycle of plants.Both biotic and abiotic stresses have certain effects on plant growth and development.In recent years,transcription factors have gradually become an important part of molecular biology research.Transcription factors can activate the expression of a series of downstream genes or participate in the regulation of various metabolic pathways to ensure the normal development of plant growth.In the previous transcriptome sequencing results,we found that a R2R3 transcription factor(StR2R3)was significantly up-regulated 24 hours after inoculation with Phytophthora infestans in the late blight high-resistant potato Jiaxiang 1 compared to control.To further study the function of the gene,plant overexpression vector was cloned and constructed,and its expression level was studied under biotic stress(P.Phytophthora),abiotic stress(drought,high salt,high temperature,low temperature),and Agrobacterium was used.The gene was transformed into Arabidopsis by immersion method.The main results are as follows: 1.Potato fluorescence quantitative reference gene screening: Real-time quantitative PCR was used to detect 10 internal reference genes ELF,EF-1β,CYP,ADF,Actin,GAPDH,E2,His,eEF,α-tubulin after treatment Jiaxiang 1 with biotic and abiotic stress respectively.The expression stability of these genes were analyzed by GeNorm,Norm Finder and BestKeeper.The expression of ELF was relatively stable after different treatments,which was suitable as the reference gene for potato gene expression detection.2.Stress-induced expression analysis: Real-time fluorescence quantitative detection of the potato transcription factor StR2R3 gene under the single stress environment as P.infestans,drought,high salt,high temperature and low temperature were carried out respectively.The results showed that the expression of StR2R3 gene was up-regulated inoculation with P.infestans after 24 h and then gradually down-regulated during 48h-72 h after inoculation.After 20% PEG treatment the expression of StR2R3 gene was first down-regulated and then significantly up-regulated.The StR2R3 gene expression was up-regulated and then downregulated under the treatment of 150mmol/L NaCl.The expression of StR2R3 genes were inhibited under high temperature at 35 ℃ and low temperature at 4 ℃.3.Gene cloning and sequence analysis: Primers were designed according to the gene sequence and the gene fragment of R2R3 transcription factor was amplified by PCR from Jiaxiang 1 DNA and named as StR2R3.The length of this gene is 958 bp which encode a 239 amino acids protein.The homology alignment and bioinformatics analysis of the protein sequence of StR2R3 with other MYB transcription factors.The result indicated that the protein contains two MYB domains,which is an R2R3-MYB transcription factor of the MYB transcription factors family.4.Construction plant overexpression vector and RNAi vector: The StR2R3 gene and its fragments were cloned into the overexpression vector pCXSN and RNAi vector PFGC5941 by restriction enzyme digestion and ligation respectively.The vectors were verified by sequencing and restriction enzyme digestion,and named as pCXSN-StR2R3 and pFGC5941-StR2R3.5.The plant overexpression vector pCXSN-StR2R3 transformate to Arabidopsis: The pCXSN-StR2R3 was transformed into Agrobacterium tumefaciens GV3101 by freeze-thaw method.The StR2R3 gene was further transformed into Arabidopsis thaliana by A.tumefaciens infection.The positive transformed plants were screened by hygromycin plate and PCR.Six Arabidopsis thaliana T3 homozygous positive plants were obtained.