| Narcissus tazetta var.chinensis is a perennial herb of the lycoris bulbous flowers and it has stout bulb,slender leaf and pleasant odor.It is a characteristic flower of Fujian province and one of the top ten famous flowers in China.It also has ornamental value and economic value.In this study,two-year and three-year-old bulbs of Narcissus were used as the experimental materials,ethylene trace phase detector(ETD-300)was employed to determine the ethylene release of bulb scale,inner scale,inner bud and basal plate through the vegetative period and dormant period.The ORF of ethylene response factor genes were cloned.The bioinformatics analysis and subcellular lacation analysis were carried out,and the expression patterns of Nt ERFs gene in the inner bud and bulb expansion and dormancy period of Narcissus were detected by q PCR.This study provides a basis for studying the mechanism of ethylene during vegetative growth and dormancy of Narcissus.The main research results were described as follows:1.The release of ethylene from the outer scales,inner scales,buds and basal plates of Narcissus was measured under different treatments during the swelling stage and dormant stage.The experimental results showed that there was not significiant change in the ethylene release from outer scales and inner scales of Narcissus at three different temperatures treatments from the end of March to the beginning of September.During the swelling stage,the ethylene release of bud at 30 ? reached a peak quickly in early April and then decreased significantly,the ethylene produced by the bulb basal plate was increasing until May when it was basically unchanged,the ethylene release of bud and basal plate at 15 ?peaked in the beginning of April and the end of April respectively,and the trend of ethylene released from basal plate and bud of Narcissus at room temperature was the same,both of which showed that ethylene production increased rapidly and reached its peak in early April and then began to decline slowly in mid-April.2.In the dormant period of Narcissus,the ethylene release of basal plate was greater than that released by bud at three temperature treatments,and the ethylene release from that at 30 ? and 15 ? showed a trend of decreasing with time from the release peak in early June.The ethylene release of basal plate and bud at room temperature showed a significant change.It appears to be a wave but not in a irregular pattern.Since early August,the ethylene release from basal plate and the bud at room temperature increased significantly,it was speculated that during this period,intense physiological activities occurred in the Narcissus,that is,the break of bud dormancy began to enter the flower bud differentiation stage and the amount of ethylene released from basal plate was significantly higher than that from other parts.The results indicating that ethylene released from basal plate may be the main factor to break dormancy induction at room temperature.After fumigation treatment of each part of the Narcissus of endogenous ethylene release a quantity to also change,one-hour-later after the fumigation measurement spend Narcissus basal plates of endogenous ethylene release quantity up to 28nl/h/g and then drops rapidly within 24 h,the other three parts of the present a slow downward trend,that fumigation processing mainly affect the bulb plate of ethylene release,also proved that the bulb plate of ethylene response plays an important role in the dormancy breaking and flower bud differentiation.3.Based on the transcriptome database of Narcissus,17 ERFs genes differentially expressed in the bulb swelling and dormant stages were screened,and 5 ERFs genes related to dormancy and growth were identified by phylogenetic tree analysis and amino acid sequence comparison.Among them,the homology of Nt ERF1 B and Arabidopsis thaliana At ERF1,At ERF14,At ERF15,Nicotiana tabacum Nt ERF1 B,Solanum lycopersicum Sl ERF4,Sl ERF-C3,Sl ERF-C7 by amino acid sequence comparison was up to 88.24%.Nt ERF1 was compared with the amino acids encoded by Nt ERF4,Nt ERF114,Nt ERF115,Nt ERF-ABR1,Nt ERF-CRF4 Arabidopsis thaliana At ERF110 and Populus trichocarpa Pt ERF119,and the homology was 65.70%.The amino acid sequence of Nt ERF2 with Arabidopsis thaliana At ERF13,At ERF1,At ERF2,Nicotiana tabacum Nt ERF1,Nt ERF2,Populus trichocarpa Pt ERF2,Solanum lycopersicum Sl ERF-A2 was 92.67%.The homology of amino acid sequences of Nt ERF114 and Nt ERF115 with Nt ERF-ABR1,Nt ERF4,Nt ERF-CRF4,Nt ERF110,Arabidopsis thaliana At ERF110 and Populus trichocarpa Pt ERF119 were 86.79% and 85.11% respectively.4.Nt ERF1 B,Nt ERF1,Nt ERF2,Nt ERF114 and Nt ERF115,five ethylene receptor genes ORF were obtained by homologous cloning with lengths of 627 bp,453 bp,576 bp,693 bp and 567 bp,and encoding 209 amino acids,151 amino acids,192 amino acids,231 amino acids and 189 amino acids respectively.Bioinformatics analysis showed that all the 5ERFs genes belonged to AP2 superfamily.The results of subcellular localization studies confirmed that Nt ERF2,Nt ERF114 and Nt ERF115 were nuclear localization proteins.5.In this study,RT-PCR was used to analyze the expression patterns of 5 ERFs in Narcissus during swelling and dormant period.And the correlation between gene expression and ethylene release were analyzed.The results showed that temperature significantly affected the expression level of ERFs,and the expression level of ERFs in different parts was also significantly different.The results showed that the expression of Nt ERF1 B in bud was positively correlated with ethylene release at 15 ?and room temperature.Nt ERF1 B and Nt ERF1 was positively correlated with ethylene release in basal plate.At room temperature,the expression of Nt ERF2 in the basal plate peaked at the end of the bulking stage and the expression of Nt ERF2 in the bud treated at 15 ? and 30 ? reached the maximum at the beginning of the dormant stage.The expression of Nt ERF2 was positively correlated with ethylene release at different temperatures.In early May,the expression level of Nt ERF114 in the bud treated at 30 ? was significantly higher than that in other parts.At room temperature,the expression level of Nt ERF114 in late may was significantly higher than that in other periods and parts.The results of correlation analysis showed that the Nt ERF114 was positively correlated with the ethylene produced by bulb at room temperature.The peak expression levels of Nt ERF115 in basal plate and bud at 30 ? and 15 ?appeared in mid-May and early June respectively.Under roomtemperature,the expression level of Nt ERF115 in basal plate significantly increased in mid-June was 139 times that of inner bud.The results of correlation analysis showed that the expression of Nt ERF115 was positively correlated with ethylene release at 15 ? and room temperature.The expression levels of 5 Nt ERFs were analyzed in basal plate and bud samples of Narcissus after fumigation treatment.The results showed that the highest expression level of Nt ERF2 in basal plate,followed by Nt ERF114,Nt ERF1 B,Nt ERF1.In summary,it was concluded that the temperature may affect the release of ethylene from Narcissus bulbs which affect the bulb entering dormancy and the release of dormancy.Ethylene released from basal plate may be an inducible factor for dormancy release of Narcissus.The Nt ERFs of basal plate and bud were involved in the ethylene signal response. |
PDF Full Text Request