Establishment Of CRISPR/Cas9 System And Study On Regulation Mechanism Of MiR2111 In Anthocyanin Accumulation Of Sweetpotato

Sweetpotato(Ipomea batatas[L.]Lam)is one of the most nutritious tuberous crops in the world.It is rich in vitamins,carotenoids,anthocyanins and other small-molecule antioxidants which are benefit for health.The purple color of purple sweetpotato tubers is mainly caused by the accumulation of anthocyanins.Anthocyanins have free radical scavenging activity that has anti-aging effect and can prevent chronic degenerative diseases.Therefore,purple sweetpotatoes are very popular among consumers.In addition to the regulation of enzyme genes and transcription factors,the biosynthesis of anthocyanins is also regulated by post-transcriptional regulator micro RNA(miRNA).In this study,the optimal reference genes for miRNA gene expression analysis in sweetpotato were selected for the first time,which provided a reliable homogenization standard for sweetpotato miRNA expression analysis.A transformation system of embryonic suspension cell and CRISPR/Cas9 system for sweetpotato were constructed.Based on multi-omics analysis of small RNA,transcriptome and degradome sequencing,the differential expression of Ib-miR2111 and its corresponding target gene IbKFB in white fleshed sweetpotato(WFSP)and purple fleshed sweetpotato(PFSP)were excavated in this study.In addition,the function of Ib-miR2111 and IbKFB in the accumulation of anthocyanin in sweetpotato were studied.The main research contents and findings were as follows:1.Optimization of reference genes for qRT-PCR analysis of miRNA expression in sweetpotatoThe expression of 8 miRNAs and two commonly used reference genes U6 and 5S in 144 samples of sweetpotato were detected by qRT-PCR.The samples included four tissues and two cultivars under three treatments.After systematic evaluation by ge Norm,Norm Finder and Best Keeper software,miRn60,miR159 and miRn60&miR159 combination were identified as the most stable reference genes under normal growth conditions,while miRn60,miR482 and miR482&miRn60 combinations were the best reference genes under stress conditions.2.Establishment of embryogenic suspension cell transformation system of sweetpotatoXushu 18(XS-18),Lizixiang(LZX),Xushu 29(XS-29),Xuzishu 3(XZS-3)and Xuzishu 8(XZS-8)were used as the receptor materials for genetic transformation.The Agrobacterium tumefaciens strains EHA105 and embryogenic suspension cells was used to establish the sweetpotato genetic transformation system.3.Construction of sweetpotato CRISPR/Cas9 systemPDS gene is a key enzyme gene in carotenoid biosynthesis of plant.The knock-out of PDS gene results in an albino phenotype,which is visible and easy to identify.In this study,we cloned IbPDS gene in sweetpotato.The full-length c DNA and genome of IbPDS were 2456 bp and 5923 bp,respectively.The fulllength genome sequence of IbPDS contained 16 exons and 15 introns.The g RNA1 and g RNA2 on the second exon of the IbPDS gene,as well as g RNA3 and g RNA4 on the 8th and 9th exons were designed,which were used to construct the vectors of CRISPR-PDS_t1/2 and CRISPR-PDS_t3/4.Transgenic sweetpotato plants with knock-out IbPDS gene were developed with A.tumefaciens-mediated transformation.Six albino phenotypic plants identified by PCR were obtained.Further sequencing results showed that the target sequence of the six albino phenotypic plants was accompanied by base substitutions or deletions.The above studies showed that the CRISPR/Cas9 system of sweetpotato were successfully established by using IbPDS gene.4.Identification Ib-miR2111 target gene IbKFBIbKFB was found to be a target gene of Ib-miR2111 by bioinformatics and degradome sequencing.The expression level of Ib-miR2111 in purple sweetpotato tubers was higher than that in non-purple potato tubers by qRT-PCR,which was consistent with the accumulation of anthocyanins.While the expression pattern of its target gene IbKFB was exactly opposite to that of Ib-miR2111,and its expression level was inconsistent with the accumulation of anthocyanin.In vivo transient expression system in tobacco leaves further confirmed that IbKFB is the target gene of Ib-miR2111.5.The function of Ib-miR2111 in anthocyanin biosynthesisThe Ib-pri-MIR2111 gene with a length of 494 bp in sweetpotato was cloned.The vector p C2300-p OT2-Ib-pri-MIR2111 of overexpressing Ib-miR2111 and STTM2111 of blocking miR2111 expressing were constructed and transformed into Agrobacterium tumefaciens strains GV3101.The T3 generation of transgenic Arabidopsis overexpressing Ib-miR2111 were obtained.Comparing with the wild-type plants,the expression level of Ib-miR2111 in transgenic Arabidopsis was significantly increased,while the expression level of At KFB was decreased.Compared with the wild-type plants,the junction between the main stem and rosette leaf of Ib-miR2111 overexpressing transgenic Arabidopsis presented deep purple color.The anthocyanin content and the expression level of related genes in anthocyanin biosynthesis pathway were also increased.The expression level of At-miR2111 in STTM2111 transgenic Arabidopsis plants was significantly decreased,while the expression level of At KFB was increased.The content of anthocyanin and the expression level of related genes in anthocyanin biosynthesis were decreased.These findings suggested that Ib-miR2111 positively regulated anthocyanin synthesis in Arabidopsis.The vector p C2300-p OT2-Ib-pri-MIR2111 were transformed into Agrobacterium tumefaciens strains EHA105 and then to embryogenic suspension cells of sweetpotato.Two positive transgenic sweetpotato plants were confirmed by PCR detection of 35S_F and Ib-pri-MIR2111_R.The results of qRT-PCR showed that the expression level of Ib-miR2111 in the two sweetpotato transgenic plants was increased,while the expression level of IbKFB was decreased.The anthocyanin content and the expression level of related genes of anthocyanin biosynthesis in the two sweetpotato transgenic plants leaves were increased.It indicated that Ib-miR2111 positively regulated anthocyanin biosynthesis in sweetpotato.Meanwhile,CRISPR-miR2111 vector was constructed and transformed into embryogenic suspension cells of sweetpotato.And six transgenic sweetpotato plants were obtained,and the target sequence of five plants had base additions,substitutions and deletions.6.The function of IbKFB in anthocyanin biosynthesisWe cloned IbKFB gene in sweetpotato.The full-length c DNA and genome sequences of IbKFB were both 1166 bp.The vector pC1300-GFP-IbKFB with IbKFB overexpressing was constructed.The subcellular localization analysis showed that IbKFB was localized in the nucleus.Compared with the wild-type plants,the color of the hypocotyl in overexpressing IbKFB transgenic Arabidopsis became lighter.The anthocyanin content and the expression level of related genes in anthocyanin biosynthesis pathway were also decreased.It indicated that IbKFB gene negatively regulated the biosynthesis of anthocyanins in Arabidopsis.The vector p C1300-GFP-IbKFB was also transformed into sweetpotato embryogenic suspension cells,which were still in the stage of inducing seedlings and no developed plants had been obtained.CRISPRIbKFB vector was constructed,and was transformed into sweetpotato embryogenic suspension cells.They were still in the stage of somatic embryo maturation.In addition,the direct interaction between IbKFB and IbPAL were found by yeast two-hybrid experiment,suggesting that IbKFB may regulate anthocyanin synthesis in sweet potato by regulating anthocyanin synthesis gene IbPAL.