Physiological, Biochemical And Molecular Mechanisms Of Fraxinus Mandshurica And Its Interspecific Hybrid F1 Entering/Releasing Dormancy

The dormancy characteristics of Fraxinus mandshurica Rupr.had important effects on the growth and stress resistance of F.mandshurica.In this study,we observed and recorded the dormancy traits and phenology of F.mandshurica and its interspecific hybrid F1 generation,and found that the interspecific hybrid F1 generation had obvious advantages in dormancy traits,such as germinating earlier and stopping growing later than the parents.We observed and recorded the different dormancy phenotypes between the female parent and the interspecific hybrid F1 generation in detail.In order to provide new ideas for genetic improvement and breeding,we further explored the formation mechanism of heterosis in this dormant trait from hormone level,physiological and biochemical indexes,gene expression and epigenetic regulation.F.mandshurica in growth period,on the other hand,the stress induced into passive dormant state resistance to bad environment,but after the environment suitable for the break their dormancy process is relatively slow,thus seriously affect its rapid growth in the growing season,here we make dormant traits according to further explore the mechanism of heterosis F.mandshurica passive dormancy rapid technology,At the same time,the key gene FmDAMs affecting dormancy was screened out to further study its specific function and influencing mechanism.The main contents are as follows:1.Differences in physiological dormancy traits between F.mandshurica and its interspecific hybrid F1 generationIn the growth cycle of one year,Maor mountain experimental forest farm of northeast forestry university for eight years living in early may enter the SAP flow of northeast China ash,then enter Meng Dong Qi about on May 14th,and complete exhibition leaf in the late may and high growth period mainly concentrated in late may to late June,after enter slow growth period and gradually formed in early July was bud scale package of the crown,However,the diameter of new shoots still increased.In the middle of September,F.mandshurica entered the leaf discoloration stage and began to defoliate from the bottom of morphology to the top of morphology.In early October,all the leaves were defoliated.However,the phenological characteristics of the F1 generation 110 combination of F.mandshurica×Fraxinus Americana Linn.,an eight-year-old interspecific hybrid of F.mandshurica in the same habitat,were different from that of the mother,mainly reflected in:（1）the germination of 110 was 7 days earlier than that of the mother,（2）the dormantic terminal bud of 110 was 14 days later than that of the mother and entered the slow growth stage,（3）the deciduous stage of 110 was 7 days later than that of the mother and both ended the deciduous stage at the same time,（4）the annual growth of new shoots of 110 was about 1.3 times that of the mother.2.Physiological and biochemical analysis of dormancy traits in F1 generation of F.mandshurica and its interspecific hybridThe results of hormone analysis showed that the differences in dormancy traits between F1 generation 110 and female parents were mainly caused by the differences in GA and ABA levels:（1）the GA level of F1 generation 110 was 1.20 times higher than that of female parents all the time before entering the deciduous stage,and decreased rapidly to similar level to that of female parents in the deciduous stage.（2）The ABA level of 110 combination was significantly lower than that of female before deciduous stage,and the lowest was about 0.89 times of that of female,but increased rapidly to similar level with that of female in deciduous stage.（3）After entering the SAP flow and germination stage,the GA level of F1 generation 110combination increased rapidly and was significantly higher than that of female parent,the highest was about 1.20 times of female parent GA content.In conclusion,higher GA and lower ABA content helped F1 generation 110 to form dormancy characteristics better than the female parent.The results of physiological index analysis showed that during the physiological dormancy period,the initial contents of soluble sugar,POD specific activity and calcium ion content in F1 generation 110 combinations were lower than or similar to that of female leaves,but the contents of soluble sugar,POD specific activity and calcium ion content in F1generation 110 combinations were significantly and rapidly increased compared with female leaves before and after leaf deciduous stage.It was about 1.27,1.31 and 1.30 times of that of the female,and it entered dormancy late but fast.In the process of releasing physiological dormancy,POD specific activity,H₂O₂ content and calcium content decreased in general,and POD decreased at a faster rate（1.07,1.64 and 1.30 times of that of the female parent）in F1generation 110 combinations to help them quickly germinate.3.Gene expression analysis and m RNA methylation analysis of dormancy traits in F1generation of F.mandshurica and its interspecific hybridThe following genes were significantly differentially expressed during entering and releasing physiological dormancy between F1 and female parents:FmSNRK and FmABI5 of ABA pathway,FmGA2ox1,FmRGA and FmGA20ox of GA pathway,FmICE1 and FmCBF of cold signaling pathway,and 6 members of dormancy related gene FmDAMs.The key dormancy gene FmDAMs（dormancy related type II Mad-box transcription factor）expressed in110 combinations was 5.4 times as high as that of the mother,and 0.31 times as high as that of the mother when dormancy was released.In the process of releasing physiological dormancy,m RNA methylation level of F1 generation 110 combination decreased faster than that of female parent（the rate was about 1.25 times）,indicating that the difference of m RNA methylation level also helped F1 generation 110 combination to release dormancy faster than that of female parent,and formed early germinating growth advantage.4.Establishment of passive dormancy removal technology for F.mandshurica:The passive dormancy of F.mandshurica under drought stress was rehydrated with GA₃treatment,methylation inhibitor（pentaazine）and Ca CN₂（lime nitrogen）treatment,respectively.The results showed that F.mandshurica could recover the growth quickly under the addition of 100 mg/LGA₃,100,200μmol/L pentaazine and 30%lime nitrogen.In the meantime,we measured the gene expression of the FmDAMs in this process and found that the expression trend of the FmDAMs was the same as that in the physiological dormancy,that is,it was rapidly down-regulated to a lower level（about 0.75 times down-regulated）to assist the dormancy release of F.mandshurica.5.Analysis of temporal-spatial expression characteristics of FmDAMs:Based on the expression of FmDAMs in the above paper,which showed a high correlation with dormancy,we further investigated the expression characteristics of FmDAMs.The results are as follows:FmDAMs are highly expressed in buds and flowers（about 10.35～99.40 times of the control）,and the lowest in stems and roots（about 0.05～0.47 times of the control）.The expression peak of The whole FmDAMs appeared in the bud elongation period on the 14th day of seedling growth after seed germination.6.Transient transformation and overexpression of FmDAM2 and FmDAM4 and their effects on gene expression of ABA/GA/cold signaling pathwayIn order to further explore the regulatory direction of FmDAM2 and FmDAM4,this paper cloned and constructed the overexpression vectors of FmDAM2 and FmDAM4.After transient infection with F.mandshurica,the GA pathway genes were mainly induced,which resulted in the significantly down-regulated expression level of FmGA20ox（GA synthesis gene）（down by0.5-0.9 times）.The expression levels of FmGA2ox1（GA catabolic gene）and FmDELLA（negatively regulated GA）were significantly upregulated（3.2 and 2.4 fold,respectively）,while the expression levels of other pathways were not the same.That is,in the process of regulating F.mandshurica dormancy,The FmDAMs mainly affect F.mandshurica dormancy state through negative regulation of GA.