Analysis Of ERF Gene Family And Establishment Of Protoplast Transient System In Dio Score A Alata L.

Dioscorea alata L.(greater yam)is the most important tuber crop after potato,cassava and sweet potato,It is also a traditional vegetable with both medicinal and edible characteristics.But is a deep-rooted crop and often grows up to one meter underground and difficult to dig.Revealing the mechnisms underneath the tuberization process of greater yam,and exploring and screening effective transcription factors play regulatory role in tuber development will provide scientific support and basis for future research on molecular breeding of greater yam.Based on the released transcriptome database and genome sequence of Dioscorea,we made systematic analysis of the ERF family genes in greater yam and identified the ERF gene related to tuber development:DaERF24.The expression profiles in different greater yam tissues of DaERF24 was detected by RT-qPCR.Moreover,we constructed an effencient isolation and transfomration system of mesophyll protoplasts in Dioscorea,and the subcellular localization of DciERF24 protein was studied.On the basis of constructing the universal CRISPR/Cas9 expression vector of Dioscorea,ERF-CRISPR/Cas9 gene knockout vector was further constructed.The main results are as below:1.Based on the published transcriptional and genomic sequences of Dioscorecr,the ERF family genes of Dioscorea were excavated and analyzed.A total of 41 DaERF genes were found,of which 14 had more than one exon and the rest were all one exon.Further blastx analysis revealed that 30 DaERF genes have conserved AP2 domains,and phylogenetic trees were constructed based on AP2 domins,which could be divided into five subfamilies.of which two conserved motifs were analyzed.It was found that ERF proteins of the same family had similar structural characteristics.The expression of DaERF in different tissues was analyzed by transcriptional profiling.It was found that the expression of some DaERF was tissue-specific and the expression of genes belonging to the same subfamily was same.DaERF24 with high expression in tubers,RT-qPCR was used to verify its expression in different tissues.The results were consistent with the transcriptional data.2.The effects of enzyme solution combination,mannitol concentration,enzymatic hydrolysis time and pH value of enzymatic hydrolysate on the yield and activity of protoplasts of dioscorea were studied by using 2-3 tender leaves as materials for enzymatic hydrolysis protoplasts in Dioscorea.The results showed that when cellulase 0.8%,dissociating enzyme 1%,mannitol 14%,0.2M MES,2M KC1,0.1%BSA,1M CaCl2 and PH 5.8,the yield and activity of protoplasts reached of the highest yield 3.70x107(g FW)and vitality 95.89%respectively.3.The concentration gradient and induction time gradient of PEG4000 were set to investigate the transformation efficiency of protoplast instantaneous expression system in Dioscorea.The results showed that when the final concentration of PEG4000 was 20%and the induction time was 20 min,the maximum conversion efficiency was 66.23%.DaERF24 and PA7 vectors were linked to construct GFP fusion expression vectors and transferred into Dioscorea protoplasts.Detection of transient expression products of DaERF24 gene showed that DaERF24 shows a strong fluorescence signal in the nucleus..4.Using DNA sequence of Dioscorea as template,we amplified the U3 promoter of Dioscorea by combining random primers(RAPD),nested PCR and promoter library,and constructed a universal CRISPR/Cas9 expression vector of Dioscorea.Which adopts pmoles ratio to 1:10 of pCAMBIA 1301-DaU3pro-2NLScas9 and 35T-BcLI’s single enzyme connection reaction,successfully build a carrier.5.The target sequence of DaERF24 gene was screened according to the DaERF24 gene sequence in the database of Dioscorea.Two knockout vectors(ERF1-A-2-CRISPR-Cas9 and ERF1-A-20-CRISPR-Cas9)of DaERF24 gene were formed after connection with the general CRISPR/Cas9 expression vector for Dioscorea in the form of joint,namely,the DaERF24-CRISPR/Cas9 gene knockout vector.After that,transforming agrobacterium EHA105 and LBA4404 fromes expression vectors.