Cloning And Characterization Of Two Novel Genes "BeSNAC1" And "BeWRKY2" From Bambusa Emeiensis And Their Transformation In Triticum Aestivum

The NAC and WRKY transcription factors(TFs)have been well characterized for their crucial role in the environmental stresses.However fewer studies were reported on NAC and WRKY TFs in Bambusa emeiensis.This is the most common species of bamboo cultivated in the Yunnan Plateau and adjacent provinces.It has been used for various purposes,from household weaving to agricultural tools,and for ornamental and soil-protection purposes.Bambusa emeiensis has a substantial ability to grow in a diverse range of habitats and successfully survive in harsh environmental conditions.The major goal of this research is the identification and characterization of unique TFs which provide Bambusa emeiensis this exclusivity and their transformation to the wheat for the development of resistant cultivar.In this research,two novel stress-responsive TFs were successfully cloned from Bambusa emeiensis,their bioinformatic analysis,expression profiling,yeast one-hybrid screening and sub-cellular localization analysis were carried out.Subsequently,their overexpression vector was constructed and transformed to the wheat genotype "DN7742" through particle bombardment.This research will provide a novel insight for the identification and characterization of unique TFs from bamboo;additionally,provide theoretical basis for the development of the resistant wheat cultivar.The research findings are subsequent:1.The genes were isolated by using the bioinformatics approaches and information from the respective databases of corresponding plants.The NAC TF was cloned by homology cloning method using the amino acid sequence of the stress-responsive SNAC1 from rice.While for the isolation of the WRKY TF the information from the genome database of the Phyllostachys heterocycla and transcriptome database of Bambusa emeiensis was utilized.The isolated TFs were named as "BeSNAC1(GenBank:MG763922)" and "BeWRKY2(GenBank:KJ462125)",respectively.The bioinformatics analysis of "BeSNAC1" and"BeWRKY2" was carried out;the findings suggest that BeSNAC1 has an open reading frame(ORF)of 912bp,which encodes a protein of 303 amino acids with a predicted molecular weight of 34.26086 kDa.Whereas BeWRKY2 has an open reading frame(ORF)of 888bp,that encodes 295 amino acid long proteins with a predicted molecular weight of 31.63580kDa.The multiple proteins Sequence alignment indicates that BeSNAC1 and BeWRKY2 protein contains a typical NAC and WRKY domain respectively.The phylogenetic analysis unveiled that BeSNAC1 belongs to SNAC group of NAC family;clade with SNAC1,HvSNAC1,TaNAC2,SbSNAC1 and ZmSNAC1 proteins,meanwhile the BeWRKY2 belongs to the group ?d of the WRKY family;clade with TaWRKY16,ZmWRKYl,ZmWRKY21,ZmWRKY74,GhWRKY30,AtWRKY7,AtWRKY11,AtWRKY15,AtWRKY17 and AtWRKY21 proteins.2.The yeast one-hybrid screening results revealed that the BeSNAC1 and BeWRKY2 both were exhibited transcriptional activity.3.Sub-cellular localization assay disclosed that BeSNAC1 and BeWRKY2 proteins were expressed in the cell nucleus.4.The results of time-dependent expression pattern analysis showed that BeSNACl and BeWRKY1 were significantly up-regulated by the ABA(Abscisic Acid),PEG(polyethylene glycol),NaCl,Na2SO4,and H2O2.The expression of BeSNACl was rapidly up-regulated by NaCI and H2O2 treatment and in response to other three treatments the up-regulation was observed during later hours,on the other hand,the expression of BeWRKY2 swiftly induced by ABA,NaCI and H2O2 treatment in other two treatments the up-regulation was delayed.The expression profiling results provide enough evidence that both of these TFs having a role in response to abiotic stresses.5.The overexpression vector was constructed and transformed to the immature callus of wheat through the particle bombardment.The successfully surviving plantlets were verified through the PCR,and qPCR were performed for the overexpression analysis.The results showed that the BeSNACl successfully transformed and overexpressed into two transgenic lines whereas for BeWRKY212 ooverexpressed lines were identified.The seeds of all transgenic plants of wheat in T0 generation were collected and planted to get T1 generation.