| Lilium lancifolium,also known as tiger lily,belongs to the family Liliaceae and the genus Lilium,which is distributed in 17 provinces of China.It is characterized by showy orange flowers and strong resistant.In addition,L.lancifolium is one of the main species in Chinese traditional edible and medicinal lilies.It’s widely cultivated in China because of its edible bulbs and medicinal uses.L.lancifolium is natural autotriploid（2n=3x=36）.As the triploids are completely sterile,and L.lancifolium therefore cannot extend its population through seeds.However,triploid L.lancifolium produces purple-black bulbils on the leaf axils along the stem.Bulbil propagation can not only retain the genetic characteristics of the parent plant,but also can be more efficient and convenient than scale-cutting and bulb breeding,making bulbil propagation a significant asexual strategy for triploid forms of L.lancifolium.At present,there is a paucity of information regarding bulbil formation.Only anatomy research has been carried out in Lilium.Although several genes related to bulbil formation have been isolated,the process of bulbil formation in L.lancifolium,the reasons why bulbils only form at the upper leaf axil and the metabolic pathways involved in the process of bulbil formation remain unclear.In this study,we used triploid L.lancifolium as materials,systematically analysing the mechanism of bulbil formation through anatomy,transcriptomics and physiology.Based on above studies,we isolated gene related to the bulbil formation,laying a solid foundation for regulating bulbil formation artificially and effectively improving the propagation coefficient of lilies.The main results are as follows:1.Morphological and anatomical observation during the formation of bulbils in L.lancifolium.We studied the rule of bulbil generation and the anatomical structure during the process of bulbil formation in L.lancifolium through morphological observation and paraffin-section method.The results indicated that bulbils of L.lancifolium formed in leaf axils,the joint region of leaf and stem,and are derived from the division and differentiation of parenchyma cells under epidermal cells at the adaxial surface of petiole base.The bulging adaxial surface of petiole base defined as bulbil initiation was the first key stage for bulbil formation of L.lancifolium.Bulbils first generated on a certain leaf axil in the upper portion of stem,and then formed upward along the upper stem.2.Physiological characteristics of bulbil formation in L.lancifolium.Soluble sugar and starch contents of upper and lower leaf axils at different stages of bulbil formation in L.lancifolium were measured using an anthrone colorimetric method.The results indicated that starch contents in upper leaf axils were higher than lower leaf axils.During bulbil initiation,soluble sugar content decreased and starch content increased.The contents of IAA,ZR,ABA and GAs were determined by means of enzyme-linked immunosorbent assay（ELISA）.The results indicated that the change of IAA content was most obvious.IAA content increased during bulbil initiation and then dropped off during the process of further bulbil formation.The contents of ZR and ABA decreased slowly during the whole process of bulbil formation.Conversely,GAs content increase slowly.3.Sequence assembly,gene functional annotation and classification.Leaf axils on the upper stem at stages S1,S2 and S3 and leaf axils on the lower stem at stage S2 were obtained for RNA-Seq using Illumina HiSeq platform.We obtained 649,714,466 raw reads.A total of 598,060,858 clean reads obtained were used in the assembly,and 389,636 contigs were generated.All contigs were assembled into 293,858 non-redundant unigenes with an average length of 549 bp and an N50 of 752 bp.The 67,672 unigenes annotated in the GO database were categorized into 56 functional groups.Based on sequence homology,36,214 unigenes were assigned to a COG functional classification,divided into 26 specific categories.According to the KEGG results,34,491 unigenes were mapped to 132 predicted metabolic pathways.4.Differential expression gene（DEGs）screening and functional enrichment analysis.In total,11,871 DEGs were identified by comparing libraries in pairs.The results of comparing libraries showed that significant differential gene expression occurred in the initiation stage of bulbil formation and the initiation process was caused by the expression of up-regulated genes.GO enrichment of DEGs showed the DEGs during bulbil formation were significantly enriched in metabolic process.The DEGs between upper and lower leaf axils were significantly enriched in catalytic activity of molecular function group and starch metabolic process,sucrose metabolic process and protein phosphorylation of biological process.KEGG enrichment of DEGs showed that pathways of starch and sucrose metabolism and plant hormone signal transduction played important roles in the process of bulbil formation in L.lancifolium.Starch synthesis promoted the initiation of upper bulbils in L.lancifolium.Hormone-associated pathways exhibited distinct patterns of change in each sample.Auxin promoted the initiation of bulbils and then inhibited further bulbil formation.5.Cloning and bioinformatics analysis of LlAGO1 gene.Based on gene fragment information of transcriptome data in L.lancifolium,AGO1 gene fragment was selected due to expression differences.According to the conserved sequence of this fragment,primers were designed,and then RACE technology was used to isolate total length of AGO1 gene,named LlAGO1.The full length cDNA of LlAGO1 was 4014 bp,which contained a 3684 bp complete open reading frame（ORF）and encoded 1227 amino acid residues with a predicted molecular weight of 135.36 kD,bioelectric point value of 9.57.In the phylogenetic tree,LlAGO1 had the closest evolutionary relationship with the homologous protein from Asparagus officinalis（XP020260210.1）.Physicochemical characteristics of LlAGO1 was analyzed and the results indicated that LlAGO1 was predicted as unstable and hydrophilic non-secretory protein,which didn’t have transmembrane structure and couldn’t form coiled coil.Amino acid sequence analysis showed that it contains two conserved domains named PAZ and PIWI.LlAGO1 was predicted to locate in the nuclear.The function of LlAGO1 was predicted as transcriptional regulation.6.Expression analysis of AGO1.The qRT-PCR analysis showed that LlAGO1 expressed in most of the tested tissues,but mainly occurred in leaf axil,and the lowest in root.In addition,the expression of LlAGO1 was characterized by its temporality and spatiality.At the different development stages of axillary bulbil,the expression of LlAGO1 gene was increased with the bulbil formation in the upper leaf axils which were able to generate bulbils,but didn’t express in the lower leaf axils which could not form bulbils. |
