Study On Genes Related To Bulb Germination Based On Transcriptome Of Narcissus Tazetta 'Huanghua 2'

Narcissus tazetta‘Huanghua 2'was identified by Fujian Crop Variety Committee in April 2012 as a new variety with special color.Narcissus tazetta‘Huanghua 2'is being spreaded on a large scale,and a large number of bulbs are urgently needed.Cutting propagation is a new technology of asexual propagation of Narcissus multiflora,which is simple,low cost and high reproductive coefficient.In this study,the bulbs of Narcissus tazetta‘Huanghua 2'in three stages of cuttage propagation:0 d,30 d and 50 d were used as materials for comparative analysis of transcriptome at different stages,and the main metabolic pathways such as starch and sucrose metabolism,cytokinin metabolism were screened and identified,and the auxin response factor family was screened and identified,and some bioinformatics was used.Methods The bioinformatics analysis of NtARF family was carried out,and the expression pattern of NtARF gene in different stages of Cuttage Propagation was analyzed by using qRT-PCR.The key enzyme gene 5'-ribose monophosphate hydrolase gene was screened and cloned from cytokinin pathway.The expression of5'-ribose monophosphate hydrolase gene in different tissues and organs during Cuttage Propagation was analyzed by qRT-PCR,aiming at exploring polychrome narcissus.The intrinsic molecular mechanism of Cuttage Propagation lays a foundation for the future use of genetic engineering technology to improve the reproductive rate of Narcissus multiflora and the development of new reproductive technology.The main results of this study are as follows:1.The original sequencing data of daffodils from 0,30 and 50 days of Cuttage Propagation were analyzed.80281762,77582098 and 68426350 Clean reads were obtained,of which Q20 and Q30 values were above 90%.167583 Unigenes were obtained from data assembly.A total of 123 798 Unigenes were annotated in 7databases of Nr,Nt,Pfam,KOG,Swiss-prot,GG and GO.There were 681up-regulated genes in Y1vsY2 group,1231 down-regulated genes in 820 and Y1vsY3groups,1942 down-regulated genes,436 up-regulated genes and 412 down-regulated genes in Y2vsY3 group.Differentially expressed genes are enriched in starch and sucrose metabolism,plant hormone signal transduction and Zeatin biosynthesis.2.Based on transcriptome data,through the annotation screening of four databases of NR,NT,Swiss-Prot and PFAM,NCBI website and online software SMART,the physicochemical properties of amino acids,protein secondary structure,subcellular localization,conservative motifs,phylogenetic trees and expression patterns of small bulbs at different stages of germination were analyzed by using ExPASy-ProtParam tool,SOPMA,Prot Comp,MEME,MAGA and Heml software.Twenty-one NtARF family genes were screened.Bioinformatics analysis showed that the length of 12full-length gene proteins ranged from 193 to 1096 aa.The predicted molecular weight was 20.46-122.2 kD and the isoelectric point was 5.19-7.97.All of them were unstable hydrophilic proteins.Subcellular localization prediction was located in the nucleus.Evolution tree and sequence analysis showed that 21 NtARF proteins were divided into three groups.Glass III was further divided into three subgroups.The ARF transcription factors with similar branches had the same or similar motifs.NtARF8,NtARF13,NtARF15,NtARF17 and NtARF21 all showed higher expression patterns,which may be through the positive regulation of auxin content to promote the germination of bulblets.3.Two LOG genes were successfully cloned from the cytokinin pathway,named NtLOG2?GenBank registration number:MK170457?and NtLOG5?GenBank registration number:MK189487?.The open reading frame?ORF?of NtLOG2 was654 bp,encoding217 amino acids,and the molecularformula was C₁₀₆₈H₁₇₀₈N₃₀₀O₃₁₇S₉,with molecular weight 24.1 kD and theoretical isoelectric point6.39.The open reading frame?ORF?of G5 is 648 bp,encoding 215 amino acids.Its molecular formula is C₁₀₅₅H₁₆₉₃N₂₉₃O₃₁₄S₉.with molecular weight 23.8kD and theoretical isoelectric point 6.08.Both NtLOG2 and NtLOG5 belong to unstable hydrophilic proteins.Transmembrane prediction analysis showed that neither protein had transmembrane structure.Through qRT-PCR analysis of the expression of two NtLOG genes in different tissues and organs at different stages of Cuttage Propagation of Narcissus tazetta‘Huanghua 2',it was found that the expression levels of two NtLOG genes were higher at 0d,30d and 50d.The expression of NtLOG2 was highest in petals at budding stage,and the highest expression of NtLOG 2 and NtLOG 5 in roots and buds was found in other tissues and organs.