Identification Of A. Comosus Var. Bracteatus LncRNAs And Functional Research Of LncABCG11

Ananas comosus var.bracteatus is an important ornamental plant with green,white and red leaves.Long-chain non-coding RNAs(lncRNAs)are a class of non-coding RNA regulatory molecules with a length greater than 200 nt,and it have not been reported in A.comosus var.bracteatus.In this study,the all-green and all-white plantlets obtained by tissue culture were used as materials.The IncRNAs in the leaves were identified by IncRNA high-throughput sequencing technology and bioinformatics analysis.The IncRNAs related to albinism in the leaves were selected through the functional enrichment of their target genes analysis.Finally,lncABCGll,lnc88969 and their target genes were cloned,and carriers of CRISPR/Cas9-lncABCG11 and pCAMBIA3301-lncABCGll were constructed to transform callus.The main findings are as follows:1.All-green plantlets(green 1,green 2,green 3)and all-white plantlets(white 1,white 2,white 3)of A.comosus var.bracteatus were used as materials.A total of 3,543 lncRNAs and 1,451 differentially expressed IncRNAs were identified by high-throughput sequencing technology.Compared with protein-coding genes,lncRNAs have lower overall expression levels,shorter sequence lengths,fewer exons,and the open reading frames(ORF)were shorten than that of mRNAs.In addition,the lncRNAs of A.comosus var.bracteatus had lower sequence conservation.2.The target genes of IncRNAs differentially expressed in all-green and all-white plantlets at three developmental stages were performed GO and KEGG enrichment analysis.GO analysis showed that the target genes of differentially expressed IncRNA were enriched in extracellular matrix,rhythm process,immune process,nutrient activity and receptor activity at three developmental stages.KEGG analysis indicated that the target genes were mainly enriched in RNA transport,ribosomes,and purine metabolism pathways during the three developmental stages.Leaf chlorophyll-related chlorophyll metabolism and photosynthesis pathways were also enriched.In the interaction analysis between IncRNAs and target genes,86 IncRNAs were found to target 36 chlorophyll metabolism-related protein-coding genes,and 25 IncRNAs and 16 target genes which were significantly differentially expressed in green-white tissues were screened.3.The sequences of IncABCGll,lnc88969 and its target gene Protoporphyrinogen oxidase(PPO)were cloned,and the sequence lengths were 861 bp,1,385 bp and 1,626 bp,respectively.Blast alignment and coding ability analysis showed that IncABCGll and Inc88969 all had no coding potential,and both were positive-sense IncRNA.Phylogenetic tree analysis showed that PPO had the highest homology with pineapple(Ananas comosus).4.Fluorescence quantification and sequencing analysis showed that IncABCGll was significantly up-regulated in albino tissue during the early stage of leaf development that the leaves were not expanded,but it was significantly down-regulated in albino tissue after green leaf significantly appeared.The expression pattern of the target gene PORB gene was the same,showing a positive regulatory relationship,and predicting its cis-acting regulation of PORB expression at the transcriptional level.The expression of Inc88969 in the first stage of all-white leaves was significantly lower than that of the all-green leaves.However,in the second and third stages after leaf development,the expression level in all-white leaves was significantly higher than that of all-green leaves.The expression pattern of the target gene PPO gene was basically the same,and it may also regulate the expression of PPO in cis mode before prediction.5.The CRISPR/Cas9-lncABCG11 knockout vector was successfully constructed transformed the callus.A total of 799 callus were inoculated by 15mg/L HygB and 20mg/L HygB for 40d.The green-white plantlets with resistant were selected and transferred to differentiation medium for 60d,then transferred to rooting medium for rooting after reaching a height of 3cm.Finally,molecular detection was performed when the root was longer than 5cm and 350 resistant plantlets were obtained.At present,180 resistant plantlets have been tested,but no specific primer bands have been detected,and positive transgenic plants have not been obtained yet.Thus,it is necessary to wait for the remaining resistant plantlets to reach the detection height and then perform selecting in the later stage.6.Through the tolerance test of different concentrations PPT(1,2,3,4,5,6,7mg/L)for callus,it was found that 6mg/L was the lethal concentration for callus.Thus,5 to 6mg/L was selected as PPT screening concentration for genetic transformation of Ananas comosus var.bracteatus.7.In this study,pCAMBIA3301-lncABCGll overexpression vector was successfully constructed and callus was transformed.A total of 664 callus were inoculated,and the green-white plantlets were obtained and resistant plantlets were selected to transfer to differentiation medium for culture by PPT 5mg/L and 6mg/L for 40d.To date,the resistant plantlets were being cultured on differentiated medium.