| Lilium is a perennial herbaceous bulbous plant belonging to Liliaceae,which is mainly distributed in temperate regions of the northern hemisphere,such as eastern Asia,Europe,North America and so on.Lilium is widely used for ornamental,medicinal and edible purposes.The lily bulblets has the characteristic of natural dormancy,and the dormancy can be changed from vegetative growth to reproductive growth after relieving dormancy at low temperature.At present,there are few reports on the molecular mechanism of dormancy release of wild lilies.In this study,wild lily bulblets were used as experimental materials,transcriptome and metabolic group sequencing and joint analysis techniques were used to explore the molecular mechanism of dormancy release of wild lily bulbs induced by low temperature.The main results are summarized as follows:(1)The construction of cryogenic transcriptome database.Wild lily bulblets treated for 0 d(CK),30d(A)and 60 d(B)at low temperature(4 ℃)were used as materials for transcriptome sequencing,and a total of 162,282 unigene were assembled.Of these,57105(35.15%)unigenes were annotated to the NR database.Compared with 30day-low temperature treatment,4,225 genes were significantly up-regulated and 3,344 genes were significantly down-regulated.Compared with 60day-low temperature treatment,3,719 genes were significantly up-regulated and 4,000 genes were significantly down-regulated after days.(2)Annotation of cryogenic transcriptome database.The starch content of wild lily bulblets decreased from 13.46g/100 g to 8.51g/100 g after low temperature treatment(4 ℃)for 30 days,and the content of soluble sugar was 2.10 times higher than that of the control;while the starch content remained at a low level after 60 days of low temperature treatment,the soluble sugar content decreased significantly.Based on transcriptome differential gene analysis,a total of 5,459 Unigenes were found to be involved in carbohydrate-related metabolism,including 57 differential genes related to starch and sucrose metabolism in lily bulbs,which were distributed in 5 pathways;the differential genes involved in glycolysis /gluconeogenesis and TCA cycle in energy metabolism pathways were mainly down-regulated,while those related to pentose phosphate pathway were up-regulated.The expression of NCED,the key enzyme in ABA synthesis pathway,was down-regulated,while the expression of GA3 OX in GA synthesis pathway was the highest at low temperature for 30 days,and the SAM in ethylene synthesis pathway was also higher than that at low temperature fo 60 days,indicating that ABA may mainly mediate dormancy maintenance of wild lily bulblets,while other plant hormones such as GA and ethylene were mainly involved in the process of dormancy release.(3)Screening of internal reference genes.Based on the transcriptome database,60 s RNA ribosomal RNA,GAPDH,EF1,Actin,UBC,TIP41,CYP,AP4,CLA and EIF were selected as candidate internal reference genes.According to the analysis by using four softwares Cq,ge Norm,Norm Finder and Bestkeeper,UBC was the most stably expressed gene in ge Norm,Normfinder and Ct analysis,while TIP41 was the most stably expressed gene in Best Keeper software analysis.(4)Analysis of metabolome of wild lily bulblets.In the metabolome database,35 metabolites were classified as amino acids,23 metabolites were classified as sugars,and 7 metabolites were classified as plant hormones.The KEGG analysis indicated that hormone metabolic pathway,amino acid metabolism,galactose metabolis etc were annotated in the low temperature metabolome.In the comparison between60 days and 0 days of low temperature treatment,the most pathways and differential metabolites were annotated,and the TCA cycle pathway was annotated,indicating that the energy metabolism in lily bulblets changed during the process of dormancy breaking after low temperature treatment.(5)Combined analysis of metabolome and transcriptome.A total of 6 metabolic pathways,19 differential metabolites and 17 differential genes were annotated by the combined analysis of transcriptome and transcriptome.Through joint analysis,D-ribose was related to SUS(sucrose synthase)and HK(hexokinase)in A-CK comparison,and amino acids were the main metabolites in B-CK comparison.It inferred that low temperature activated the metabolic reactions in wild lily bulbs,which served the bud growth.In B-A,genes,such as IAA14 in auxin biosynthesis pathway,SIMKK in ethylene pathway,PYR1 and PP2 C in ABA pathway,all participated in 4-hydroxyindole synthesis,indicating that hormone-regulation is a complex pathway. |
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