Cloning And Functional Analysis Of MiR172 Gene Family And Its Target Gene IbAP2 In Sweet Potato

Sweet potato belongs to the dicotyledonous plant of Convolvulaceae,which has high yield,comprehensive nutrition and rich in amino acids,vitamins,protein,anthocyanins and other nutrients,so it is recommended as the best food by the World Health Organization.The miRNA is a ubiquitous,endogenous,non-coding single-stranded RNA,in organisms,which plays an important role in regulating many biological processes,such as growth and development,morphogenesis,stress response and so on.The miR172 is a highly conservative miRNA.The target genes of miR172 are AP2/ERF family genes.AP2/ERF is a transcription factor that exists widely in plants,which is closely related to plant growth and development and stress.miR172 regulates floral organ development,flowering time,fruit development and stress by acting on AP2.In this study,we analyzed the miR172 gene family from the whole genome level of sweet potato for the first time,cloned Ib-miR172 and its target gene IbAP2;,analyzed the expression of Ib-miR172 and its target gene IbAP2 in different sweet potato varieties and different tissues,constructed the overexpression vectors of p C2300-p OT2-Ib-pri-miR172 and p C1300-IbAP2-GFP,and verified that IbAP2 is the target gene of Ib-miR172.The main results are as follows:1.Ib-miR172 gene family.There are three members of sweet potato miR172 gene family,and the precursor sequences of the three miR172 genes can form a stable stem-loop structure.Ib-miR172 a and Ib-miR172 b are distributed on chromosome 3 and Ib-miR172 c on chromosome 11.In terms of evolutionary relationship,Ib-miR172 a and Ib-miR172 b are clustered on the same evolutionary branch.There are mainly 12 cis-acting elements in the promoter sequence of Ib-miR172 gene,including abiotic stress response elements such as drought and low temperature.A total of 20 miR172 target genes were obtained by prediction,including the AP2 gene family.2.Cloning,sequence Analysis and Vector Construction of Ib-pri-miR172 and its Target Gene IbAP2 from Sweet Potato.Ib-pri-miR172,was cloned from "Xuzishu 3" and "Xushu 18" respectively,and its size was 421 bp.Using MEGA7.Based on the multi-sequence alignment analysis between Ib-miR172 and other plant miR172 mature sequences,it was found that miR172 was highly conservative and the p C2300-p OT2-Ib-pri-miR172 overexpression vector was constructed.The IbAP2 gene was cloned from "Xuzishu 3" and "Xushu 18".Its ORF is 1395 bp,encoding 464 amino acids.There are two AP2 domains in the amino acid sequence,and there is a YRG conserved motif in each domain,which belongs to the AP2(APETALA2)subfamily of AP2/ERF transcription factors.The full-length DNA of IbAP2 is 3185 bp and contains 10 exons and 9 introns.The AP2 transcription factor is a basic unstable hydrophilic protein without signal peptide and transmembrane domain.The tertiary structure showed that the protein had three β-folds and one α-helix structure.In terms of evolutionary relationship,IbAP2 has the closest genetic relationship with three-leaf split potato.The promoter sequence of IbAP2 contains a variety of cis-acting elements of transcription factor binding sites such as MYB and MYC.The overexpression vector p C1300-IbAP2-GFP of IbAP2 was constructed.3.Expression Analysis of Ib-miR172 and its Target Gene IbAP2The expression of Ib-miR172 and its target gene IbAP2 in different tissues of sweet potato and different white and purple heart sweet potatoes were analyzed by q RT-PCR.The transient expression test of tobacco proved that IbAP2 was the target gene of Ib-miR172.To sum up,this experiment analyzed the miR172 gene family from the whole genome level of sweet potato,and clarified the distribution of Ib-miR172 in the sweet potato genome and its position in the evolutionary relationship.The expression of Ib-miR172 and its target gene IbAP2 in different tissues and varieties of sweet potato was analyzed by q RT-PCR,and the transient expression of IbAP2 gene in tobacco was verified to be the target gene of Ib-miR172.Ib-pri-miR172 and its target gene IbAP2 were cloned and overexpression vectors p C2300-p OT2-Ib-pri-miR172 and p C1300-IbAP2-GFP,were constructed,which laid a foundation for further study on the biological function of Ib-miR172 and its target gene IbAP2.