| In this study,the Fraxinus Interspecific hybrids whose male parents were F.americana Linn.and F.sogdiana Bunge from Beijing and Xinjiang and F.velutina Torr.from Tianjin and Panjin,respectively,and female parents were F.mandschurica Rupr.from Harbin and Dailing,respectively,were used as the main object of study.To select the superior hybrid combinations,the growth traits of 126 different Fraxzinus Interspecific hybrids and 37 random pollination progeny of their parents were analyzed;to study the mechanism of drought-advantage in hybrids,the drought treatment was conducted with some hybrid combinations;to study the circadian clock regulation,two circadian genes,LHY and TOC7,were cloned and their rhythmic expressions were analyzed,furthermore,the differences and correlations between the expressions of circadian genes and ABA-related genes under drought condition were analyzed;to further research the molecular mechanism for the responses of hybrids and their female parent against the drought condition,iTRAQ protein quantitative analysis technique was conducted.This research provided a new idea for revealing drought-advantage mechanism in Fraxinus interspecific hybrid,and laid the foundation for the researches of drought-related proteins and its coding genes,as well as the cultivation of the fast growing and drought resistant hybrids.1.The selection of superior Fraxinus Interspecific hybrid combinations(1)The growth traits analysis of Fraxinus Interspecific hybrids:the differences of Height,Diameter at breast height,Base diameter and Volume of wood between hybrid combinations were very significant.The Height,Diameter at breast height,Base diameter and Volume of wood of each line were significantly higher than their parent,among these the Fraxinus mand.shurica × Fraxinus velutina were the highest,increased 12.3%?14.5%?7.4%and 38.7%compared to Fraxinus mundshurica,respectively,and increased 22.2%?37.3%?52.2%and 108.1%compared to Fraxinus velutina,respectively.(2)The selection of superior Fraxinus Interspecific hybrid combinations:In this study,the Diameter at breast height as the basis for selecting between Fraxinus species hybrids excellent pedigree,according to the super-parent heterosis more than 25%,the mean diameter exceeds the overall average diameter of Fraxinus interspecific hybrids more than 20%as a standard.initially selected 12 excellent hybrids,according to the Diameter at breast height descending order:D104?D105?D119?D116?D103?D78?D110?D95?D66?D94?D83 and D67.2.The physiological responses of Fraxinus hybrids under drought stress:there were different degrees of inhibition in growth and photosynthesis between each hybrid combinations after 12 days under drought condition with 25%relative moisture content,meanwhile the MDA contents and Antioxidant Enzyme Activity increased in different degrees.After rehydration,the majority of physiological indexes recovered.The ABA content of each hybrids increased compared to control under drought stress,GA content reduced,similar trends in IAA and ZR contents with ABA content,after rehydration,endogenous hormone content of some plants recovered.The drought resistant hybrids D110 and D16 maintained higher growth traits,photosynthesis and Antioxidant Enzyme Activity compared to their female parents,as well as lower MDA contents,and accumulate more ABA.IAA and ZR hormones and fewer GA hormone compared to their female parents.3.The analysis of circadian genes LHY and TOC1.(1)The length of LHY cDNA was 2448bp,and its homology with the grapes reach 73%,it contained an ORF of 2313bp which encoding 770 Aa.The homology with sesame seeds and grape LHY gene encoding protein sequence were more than 60%,the evolutionary analysis showed a close relationship with sesame seeds,and farthest genetic relationship with Arabidopsis;The length of TOC1 cDNA was 2080bp,and its homology with the sesame seeds reach 78%,it contained an ORF of 1581 bp which encoding 526 Aa.The homology with sesame seeds and grape LHY gene encoding protein sequence were more than 60%,the evolutionary analysis showed a close relationship with sesame seeds,and farthest genetic relationship with Arabidopsis;(2)The expression of circadian gene LHY and TOC1 in ash leaves changed within the day as time showing generate circadian fluctuations.The expressions of LHY were at a high level between ZTO to ZT6,and a low level between ZT9 to ZT21,while the expressions of TOC1 were at a high level between ZT15 to ZT21,and a low level between ZTO to ZT12,indicated there was a one after another,mutual inhibition effect between the expression of LHY and TOC1;4.The analysis of molecular mechanisms for drought-advantage in Fraxinus Interspecific hybrids:(1)The responses of circadian gene expression under drought condition:the expression of TOC1 in hybrids down-regulated under drought condition,the decrease ratios ranged from 20.9%to 60.1%,and the expression of TOC1 in hybrids were lower than the mean parent value(MPV)of their parents,the decrease ratio ranged from 10.2%to 37.8%;in the contrast,the expression of LHY in hybrids up-regulated under drought condition,the increase ratios ranged from 32.7%to 93.5%,and the expression of LHY in hybrids were higher than the mean parent value(MPV)of their parents,the increase ratio ranged from 22.8%to 90.2%.The down-regulation of TOC1 and up-regulation of LHY were greater in hybrids than parents,indicated that changing the amplitude of circadian gene expression in hybrids under drought condition might be a possible molecular mechanism for drought-advantage in hybrids;(2)The responses of ABA-related gene expression under drought condition:the expressions of NCED?PYR1 and SnRK2.6 in the drought resistant hybrids D110 and D16 up-regulated under drought condition,the increase ratios were higher than their female,and were 108.3%and 54.0%,74.2%and 167.3%,56.1%and 63.8%,respectively;meanwhile the expressions of NCED,PYR1 and SnRK2.6 in the poor drought resistant hybrids D15 and D47 down-regulated under drought condition,the decrease ratios were 35.0%and 34.0%,43.3%and 81.3%,8.2%and-10.3%.furthermore,the PYR1 and SnRK2.6 expression level of D110 and D16 were higher than their female parents,among these the PYR1 and SnRK2.6 expressions of D110 were 1.9-fold and 1.1-fold compared to its female parent respectively,and he PYR1 and SnRK2.6 expressions of D16 were 6.5-fold and 2.0-fold compared to its female parent.Combined with the physiological response of each hybrid under drought stress,the differential expressions of these ABA-related genes between different hybrid combinations might be a possible molecular mechanism for drought-advantage in hybrids,these more sensitive responses of NCED,PYR1 and SnRK2.6 expressions in hybrid D110 and D16 compared to their parents and other hybrids,along with the higher levels of PYR1 and SnRK2.6 expressions might coursed the fine drought physiological responses.5.The proteomic analysis of drought resistant hybrid combination under drought condition:(1)The protein quantitative analysis of Fraxinus Interspecific hybrid:a total number of 4927 proteins were identified by using iTRAQ technique,the number of differential expressed proteins in drought resistant hybrid D110 under drought condition was 1145,among these 543 proteins were up-regulated in control compared to treatment,and 602 proteins were down-regulated in control compared to treatment.There were 1228 differential expressed proteins between drought resistant hybrid D110 and its female parent D113,among these 637 proteins were up-regulated in female parent D113 compared to hybrid D110,and 591 proteins were down-regulated in female parent D113 compared to hybrid D110.(2)The correlation analysis of the differential expressed proteins in Fraxinus Interspecific hybrid:to study the differential expression of drought-related proteins between hybrid and its female parent,the correlation analysis of these two groups of differential expressed proteins was conducted,result showed that there were 588 proteins differential expressed in both comparisons which clustered into 4 different expression patterns,and the expression levels of most differential expressed proteins were higher in hybrid than female parent when these differential expressed proteins were up-regulated under drought condition;in contrast,the expression levels of most differential expressed proteins were lower in hybrid than female parent when these differential expressed proteins were down-regulated under drought condition.The result above showed that the responses of protein expression regulations were greater in drought resistant hybrid than its female parent.(3)The KEGG pathway analysis of the differential expressed proteins in Fraxinus Interspecific hybrid was conducted,result showed that the differential expressed proteins under drought condition between drought resistant hybrid and its female parent mainly related to some Pathways,including Metabolic pathways,Biosynthesis of secondary metabolites,Glycolysis/Gluconeogenesis,Ribosome,Protein processing in endoplasmic reticulum,Oxidative phosphorylation,Phenylpropanoid biosynthesis,Carbon fixation in photosynthetic organisms,Plant-pathogen interaction,RNA transport and Plant hormone signal transduction and so on.In short,the molecular mechanism for drought-advantage in Fraxinus Interspecific hybrid might generated from:the changing responses of circadian genes expressions in hybrids compared to their parents,inactivated the expressions of downstream genes including ABA signal transduction related genes,further regulated the proteins related to Pathways including Metabolic pathways,Biosynthesis of secondary metabolites,Glycolysis/Gluconeogenesis,and Protein processing in endoplasmic reticulum,led to the differential expressions of these proteins between hybrid and its parents,resulted in the different physiological responses between hybrids and their parent,generated the drought-advantage in Fraxinus Interspecific hybrids. |
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