Identification And Functional Characterization Of MiR2111?miR156 And Their Corresponding Targets In Sweetpotato

The tuberous root of purple-fleshed sweetpotato(PFSP)is anthocyanins riched,which have healthy effects on increasing body immunity,anti-cancer,lowering blood sugar and cardiovascular protection.Micro RNAs(mi RNAs)are endogenous non-coding small RNAs,which play important roles in biological processes,such as plant growth and development,stress responses,and secondary metabolites.At present,the mechanism of the role of mi RNAs in the biosynthesis of the tuberous roots of PFSP is still unclear.In the early stage of the study,multi-omics analysis of the transcriptome,small RNA sequencing and degradome showed that 121 mi RNAs were differentially expressed between PFSP and white-fleshed sweetpotato(WFSP).Among the 121 mi RNAs,26 mi RNAs and their corresponding 36 target genes potentially participated in the biosynthesis of sweetpotato anthocyanins.This study mainly focus on the functions of Ib-mi R2111,Ib-mi R156 and targets genes in anthocyanins biosynthesis in sweetpotato.The results are as following:1.Cloning and expression of Ib-pri-MIR2111 from sweetpotato.A length of 355 bp Ib-pri-MIR2111 was obtain by PCR and sequencing.Degradome sequencing and q RT-PCR analysis showed that the target gene of Ib-mi R2111 is Ib ZFP(zinc finger protein).The ORF length of Ib ZFP were 1290 bp,which encoding 429 amino acids with Lab A_Like and zinc finger domains.Ib ZFP belongs to the C2-1i C type according to the C2H2 zinc finger structure.Quantitative real-time PCR(q RT-PCR)analysis showed that the expression of Ib-mi R2111 was higher in PFSP than in WFSP.On the contrary,the expression pattern of Ib ZFP showed opposite trend with that of Ib-mi R2111.The expression of different tissues showed that Ib-mi R2111 had the highest expression in the roots and the lowest expression in the leaves,which was opposite to the expression pattern of Ib ZFP in the corresponding tissues,suggesting that the expression of Ib ZFP was regulated by Ib-mi R2111.Furthermore,the overall expression level of Ib-mi R2111 in the above-ground organs of WFSP was significantly higher than that of PFSP.On the other hand,the expression of Ib-mi R2111 in the under-ground organs of WFSP was significantly lower than that of PFSP.Whether in the above-ground organs or in the under-ground organs,the expression pattern of Ib ZFP showed opposite trend with that of Ib-mi R2111 between WFSP and PFSP,suggesting that Ib-mi R2111-Ib ZFP mediated anthocyanin accumulation may have a transfer mechanism between above-ground and under-ground organs in sweetpotato.2.Function of Ib-pri-MIR2111.The Ib-pri-MIR2111 and target gene Ib ZFP were cloned into the corresponding site of the p CAMBIA2300 to construct the plant expression vectors p C2300-p OT2-Ib-pri-MIR2111 and p C1300-Ib ZFP,respectively.The Ib-pri-mi R2111 and Ib ZFP CRISPR/Cas9 plant expression vector was constructed.Four anthocyanin pathway genes(At CHS,At CHI,At DFR,At ANS)showed upregulated expression of was detected in ib-mi R2111 over-expressing Arabidopsis plants.Compared with the wild type,the bottom of the main stem of the transgenic plants showed a purple color,indicating that Ib-mi R2111 plays a positive regulatory role in anthocyanin synthesis.3.Cloning and expression of Ib-pri-MIR156 from sweetpotato.A length of 615 bp Ib-pri-MIR156 was obtain by PCR and sequencing..Degradome sequencing and q RT-PCR analysis revealed that the target gene of Ib-mi R156 is Ib SPL2(SQUAMOSA promoter binding protein-like 2).The DNA length of Ib SPL2 is 2960 bp,which have four exons and three introns.The ORF length of Ib SPL2 is 1269 bp,encoding 422 amino acids with SBP conserved domain.q RT-PCR analysis showed that the expression of Ib-mi R156 was significantly higher in PSFP than in WFSP.On the contrary,the expression pattern of Ib SPL2 showed opposite trend with that of Ib-mi R156..The expression of different tissues showed Ib-mi R156 had higher expression in leaves and petiole,while Ib SPL2 has the highest expression in roots.The expression of tuberous root during different developmental stages indicated that Ib-mi R156 has the highest expression in the 90-day tuberous roots compared with other periods,while Ib SPL2 has the lowsest expression in 90-day tuberous roots.The negative patten between the expression of Ib SPL2 and Ib-mi R156 suggested that Ib SPL2 was regulated by Ib-mi R156.The expression of sweetpotato seedlings at the "five-leaf-one-heart" stage under phosphorus deficiency showed that both Ib-mi R156 and Ib SPL2 were induced by phosphorus deficiency.The Ib-pri-MIR156 and Ib SPL2 overexpressing vectors were constructed.The ib-mi R156 over-expressing transgenic Arabidopsis showed upregulated expression of four anthocyanin pathway genes(At CHS,At CHI,At DFR,At ANS).Compared with.wild-type Arabidopsis,the stem and young leaves of the transgenic plants showed purple color,indicating that Ib-mi R156 positively regulates the biosynthesis of anthocyanins.4.The SPL gene family was analyzed and identified by using the I.triloba genome,which is a diploid wild relative of sweetpotato.According to the classification criteria of the Arabidopsis At SPL gene family,the 26 Itb SPL family genes were divided into 7 groups.SPL genes from I.triloba and Arabidopsis were grouped together,suggesting a similar function of SPL genes.Ps RNA target prediction and q RT-PCR assay analysis found that 13 of Itb SPL are target genes of mi R156.5.The regeneration system of embryogenic suspension cells of sweetpotato has been successfully established,and the p C2300-p OT2-Ib-pri-MIR2111 vector was transformed into calligenic embryos.In summary,Ib-mi R2111 and Ib-mi R156 were cloned in sweetpotato for the first time,and their target genes were identified as Ib ZFP and Ib SPL2,respectively.The Ib-mi R2111 and Ib-mi R156 over-expressing transgenic Arabidopsis demonstrated that Ib-mi R2111 and Ib-mi R156 positively regulated the biosynthesis of anthocyanins.The above results enriched the regulation mechanism of plant anthocyanin biosynthesis,particularly in tuberous root of sweetpotato,and also provide new breeding targets for regulating plant anthocyanin content through genetic engineering.