MicroRNAs Identification And Screening And Functional Research Of Key MicroRNAs Involved In The Albino Of Ananas Comosus Var. Bracteatus

Ananas comosus var.bracteatus has important ornamental value due to its unique colorful chimera leaves.It is an important new ornamental plant.Our research group has screened out the key structural genes leading to leaf albino,and continues to explore the molecular mechanism of its leaf albino from the perspective of microRNA(miRNA),which can further clarify the molecular mechanism of green-white chimeric trait formation.In this paper,complete white(CWh)and complete green(CGr)leaves in the three developmental stages of Ananas comosus var.bracteatus were used as the typical materials,and high-throughput sequencing of small RNA was carried out.The miRNAs in Ananas comosus var.bracteatus were identified,and the target genes were predicted and functionally annotated.Combined with the differential expression analysis of miRNA and functional annotation of target genes,the key miRNAs of albino were screened,and the miRNA overexpression vector was used to genetically transform the Ananas comosus var.bracteatus to verify its function.The main findings obtained are as follows:1.The content of chlorophyll and carotenoids in CWh and CGr leaves were determined.The results showed that chlorophyll was not detected CWh leaves at all developmental stages,and the chlorophyll content in CGr leaves increased with leaf development and growth.It is proved that CWh and CGr leaves are typical materials of mutant albino cells and normal green cells.The carotenoid content of CWh leaves was also significantly lower than that of CGr leaves,and the difference was more significant after the leaves were unfolded.2.163 novel miRNAs were identified from Ananas comosus var.bracteatus by high-throughput sequencing of small RNA.Among them,104 miRNAs were conserved between species,and 59 miRNAs were not conserved.Of the 163 miRNAs,123 miRNAs predicted 488 potential target genes.Of these,414 target genes were annotated into the Nr,Nt,Swiss-Prot,GO,COG,KEGG,KOG and Pfam databases.3.The expression profile of miRNA during the development of CWh and CGr leaves was constructed.When the leaves were not stretched,the green of the CGr leaves was not obvious,and there were only 29 miRNAs differentially expressed between the CWh and CGr leaves.The miRNAs differentially expressed between the CWh and CGr leaves during the second and third developmental stages after the leaves were unfolded.There are65 and 69 miRNAs differentially expressed between the CWh and CGr leaves.From the development of CWh and CGr leaves to the maturity of the leaves,the number of differentially expressed miRNAs decreased from 80 and 91 to 69 and 53 respectively.And during the second to third stages of leaf development,the number of down-regulated miRNA genes is approximately 7-fold and 5-fold higher than the number of miRNA genes up-regulated.4.The expression levels of nine differentially expressed miRNAs and their target genes were detected by qRT-PCR.The results showed that the expression trends of miRNAs were consistent with the sequencing results,and the reliability of sequencing results was verified.The expression relationship between miRNA and its target gene was analyzed.The results showed that the expression levels of these target genes are not all dependent on their predicted miRNAs and showing a negative regulatory relationship.5.Combining differential expression patterns of miRNAs between the CWh and CGr leaves and functional analysis of their target genes,7 key miRNAs involved in the albino of Ananas comosus var.bracteatus were selected: Ab-miR127,Ab-miR160,Ab-miR161,Ab-miR163,Ab-miR6,Ab-miR7 and Ab-miR8.Ab-miR127,Ab-miR160,Ab-miR161 and Ab-miR163 co-target the divinyl chlorophyllide a 8-vinyl-reductase gene(DVR),Ab-miR6,Ab-miR7 and Ab-miR8 target PGR5-like protein,which is involved in the cyclic electron fow(CEF)around photosystem I.6.The precursor sequences of Ab-miR127,Ab-miR160,Ab-miR161 and Ab-miR163 and the full-length sequence of its target gene AbDVR were cloned and sequenced.The results showed that pre-Ab-miR127 lacked the 11 bp miRNA mature sequence and was a false positive result of sequencing prediction;pre-Ab-miR160/161 had a deletion of about 60 bp,but the mature miRNA sequence was intact;The homology of pre-Ab-miR163 and Ananas comosus homologous sequence was 100%,so pre-Ab-miR163 was selected for functional verification.The cloned AbDVR gene contains a 1236 bp open reading frame(ORF)encoding 411 amino acids,and its nucleic acid sequence is 100% homologous to AcDVR.7.In order to determine the culture conditions of hygromycin from the genetic transformation of red peony pineapple,the tolerance test of callus to hygromycin wascarried out.It was found that 30mg/L is the lethal concentration of callus of Ananas comosus var.bracteatus,and 25-30 mg/L is the HygB screening concentration suitable for the callus of Ananas comosus var.bracteatus.8.The UBi-pCAMBIA1301-pre-AbmiR163 overexpression vector was successfully constructed and transformed into callus of Ananas comosus var.bracteatus.A total of 3batches were transformed,and 892 calli were transformed.After screening with 25mg/L HygB and 30mg/L HygB for 40 d,43 resistant calli were obtained by green and white seedling screening.However,the resistant shoots grew slowly.After 90 days of differentiation,only 11 resistant shoots had a plant height of 2-3 cm.The conversion period is approximately 200 days.Later,it is necessary to wait for the resistant plants to reach the detection height and then verify.