Isolation And Identification Of Related Gene For Leaf Spines Formation In Ananas Comosus

Ananas comosus is a perennial herbaceous fruit tree.It is a special economic crop in the tropics and one of the important tropical fruits in the world.The leaf edge of pineapple is often spiny,which leads to a lot of inconvenience in the process of cultivation and field management.The cultivation of spineless varieties is one of the important objectives of pineapple breeding.Using pineapple Tainong 16 as a test material,seven genes related to leaf spines formation in pineapple were obtained based on transcriptome sequencing data.They are AcBLH2(Ananas comosus BEL1-like homeodomain protein 2),AcLBD15(Ananas comosus LOB domain-containing protein 15),AcKNOX1(Ananas comosus homeotic protein knotted-1-like),AcSPL14(Ananas comosus squamosa promoter-binding-like protein 14),AcTBL19(Ananas comosus protein trichome birefringence-like 19),AcWATl(Ananas comosus protein WALLS ARE THIN 1)and AcWOX3(Ananas comosus WUSCHEL-related homeobox 3-like).Bioinformatics analysis and expression pattern analysis of the above target genes were performed.The expression vector was constructed and transformed into model plant Arabidopsis thaliana for functional identification.The role of the genes was preliminarily elucidated for leaf spines formation in pineapple.The main findings are as follows:1.The full-length cDNA sequences of 7 genes for leaf spines formation were successfully isolated from the spiny leaf edge of pineapple and spineless leaf edge of pineapple.The results showed that there was almost no difference in the sequence of these seven genes in different leaf margin.They were named AcBLH2,AcLBD15,AcKNOX1,AcSPL14,AcTBL19,AcWATl and AcWOX3,respectively according to the predicted sequence names on the NCBI website.2.Quantitative real time PCR(qRT-PCR)analysis showed that there were 5 genes(AcBLH2,AcLBD15,AcKNOX1,AcSPL14 and AcTBL19)differentially expressed in different leaf margin of pineapple.There were 2 genes(AcWAT1 and AcWOX3)with no significant difference.The relative expression levels of AcBLH2,AcLBD15 and AcTBL19 genes in the spineless leaf edge of two varieties of pineapple were significantly higher than those of the spiny-leaf pineapple,while the relative expression levels of AcKNOX1 and AcSPL14 genes in the spineless leaf edge of two varieties of pineapple were significantly lower than those of the spiny-leaf pineapple.The differences of the expression levels of these five genes in different leaf edges of two cultivars were consistent,indicating that the variety difference had no effect on the expression of the five genes in different leaf margin of pineapple.In addition,the time-space-specific expression of the 7 genes related to leaf spines formation was analyzed by qRT-PCR analysis.Except for the highest expression of AcBLH2 gene in pineapple leaves,the other 6 genes were highest expressed in pineapple stem.3.The domain analysis of the proteins encoded by the five genes showed that all of the five genes had conserved domains of the their corresponding gene families,and their functions were conservative.Phylogenetic tree analysis showed that there were no species closely related to pineapple in the BLH,SPL14 gene family;Jujuba is closely related to pineapple in KNOX1,LBD15,TBL19 gene family.4.Five over-expression vectors of genes related to leaf spines formation were successfully constructed and transformed into A.thaliana.All the positive plants were obtained.There was no significant difference between 35S::AcBLH2,35S::AcLBD15,35S::AcSPL14 and 35S::AcTBL19 positive plants and wild type of A.thaliana.The leaves of A.thaliana transformed with AcKNOX1 were crimping and spliting deeper and leaf epidermal hair increased significantly.The leaves of A.thaliana showed etiolation at the later stage of growth and development.5.The subcellular localization experiment of onion inner epidermis cells showed that AcKNOX1 was localized in the nucleus of onion,which further confirmed AcKNOX1 playing a regulatory role in nucleus as a transcription factor.6.The promoter of AcKNOX1 gene was successfully obtained from the spiny leaf edge of pineapple and spineless leaf edge of pineapple.Sequencing results showed that there was no significant difference in nucleic acid sequence between the two promoters.Analysis of the promoter bioinformatics showed that there were several hormone response elements,such as gibberellin,cytokinin and abscisic acid,which might regulate the expression of genes related to hormone synthesis pathway(response pathway)and affect plant growth and development.The promoter of AcKNOX1 also has some other regulatory elements,such as photoresponse element,anaerobically induced cis-regulatory element and environmental stress response element,indicating that AcKNOX1 gene may also be involved in other biological pathways.Three hormones,gibberellin,cytokinin and abscisic acid,were used to treat pineapple seedlings.The expression of AcKNOX1 in pineapple seedlings increased at first and then decreased,and the peak at 8 h,12 h and 12 h,respectively.The results showed that all three hormones could induce the expression of AcKNOX1 gene.In this study,7 genes for leaf spines formation in pineapple were successfully isolated from pineapple leaves with different leaf edge morphology.Only AcKNOX1 gene may play a key role in the occurrence of pineapple leaf thorns.In spiny and leafless pineapple leaves,the differential expression of AcKNOX1 gene was not caused by the difference of promoter sequence.And exogenous hormone treatment may induce the increase of AcKNOX1 gene expression,which may lead to the occurrence of pineapple leaf thorn.This study are helpful to understand the regulation mechanism of pineapple leaf shape development and lay a foundation for the cultivation of spineless-leaf pineapple varieties.