| Fraxsinus mandshurica Rupr,As an important precious large-diameter timber tree species in Northeast China,it has an important position and wide application.In this study,the growth and cold resistance characteristics of Fraxinus mandshurica x Fraxinus Fraxinus hybrid F1 aged 4～14 years in Maoershan,dahelin,Linjiang and dailing were investigated,the growth and cold resistance advantages of 35 hybrid combinations of 5 families were analyzed,and excellent hybrid combinations with excellent growth and cold resistance were selected.The formation mechanism of cold resistance Heterosis of Hybrid F1 was further explored through the analysis of survival rate,cold resistance index,physiology,biochemistry and gene expression.The main results are as follows:1.Interspecific hybrid of Fraxinus mandshurica(Fraxinus mandshurica)×Selection of excellent germplasm resources of Fraxinus tomentosa:through the data investigation and statistical analysis of adaptability and growth traits of Hybrid F1 in four test sites,the comprehensive selection of adaptability,growth traits and cold resistance traits was carried out.The results showed that:(1)Adaptive(survival rate)selection:there are 10 hybrid combinations such as 108,116 and 117 whose survival rate exceeds the total average by more than 15%(the variation range is 15.91%～33.62%,and the average value is 24.63%),among which the 108 hybrid combination with the highest survival rate exceeds the total average and the female parent control by 33.62%and 19.2%respectively,indicating that 10 hybrid combinations such as 108 and 116 have obvious heterosis in survival rate,And 108 hybrid combination is the most adaptable.(2)Selection of growth traits:there were 11 hybrid combinations 108,83,117,96,95,116,120,111,103,119 and 112 whose growth traits exceeded the total mean value by more than 15%(the range of variation was 20.44%～158.04%,and the mean value was 57.33%),among which the hybrid combination 108 had the most obvious heterosis of growth traits,exceeding 55.13%of the female parent control and 149.8%of the total mean value.It shows that 11 hybrid combinations such as 108 and 83 have obvious heterosis in growth characters,and 108 is the best hybrid combination.(3)Selection of cold resistance traits:there were 9 hybrid combinations 108,96,122,120,103,94,79,129 and 82 whose cold resistance traits exceeded the total mean value by more than 15%(the range of variation was 15.46%～64.77%,and the mean value was 33.99%).The heterosis of cold resistance traits of hybrid combination 108 was the most obvious,275.5%higher than that of female parent control and 64.8%higher than the total mean value.It shows that the Heterosis of 9 hybrid combinations such as 108,96 and 122 is the most obvious in cold resistance characters,and 108 is the best hybrid combination with cold resistance.To sum up,two excellent hybrid combinations 108 and 103 with excellent adaptability,growth and cold resistance were selected,of which 108 was the best hybrid combination.2.Study on the formation mechanism of cold resistance Heterosis of hybrid combinations:taking dahelin test site as the representative,take the leaves for artificial simulated cold treatment(-5℃,24 hours),measure the physiological and biochemical indexes and related gene expression,and analyze the formation mechanism of cold resistance Heterosis of excellent F1 generation.The results are as follows:(1)Physiological and biochemical analysis of Fraxinus mandshurica and its interspecific hybrid defense system under cold stress:under cold stress,the overall physiological and biochemical indexes of Hybrid F1 were stronger than those of female parent control,and the relative conductivity and MDA content(membrane system)of Hybrid F1 were lower than those of female parent control;The contents of soluble sugar and soluble protein(osmotic system)in Hybrid F1 were higher than those in female parent control;The activities of pod and SOD enzymes(ROS scavenging system)in Hybrid F1 were higher than those in female parent control.It is speculated that the physiological and biochemical changes of defense system lead to cold resistant heterosis in excellent F1 generation.(2)Differential expression analysis of cold resistance related genes in Fraxinus mandshurica and its interspecific hybrids:in the rhythm gene nac-dreb/cbf-cor gene pathway under cold stress,the expression of key genes in Hybrid F1 is higher than that in female parent control,among which hybrid combination 108 is the most prominent,and the expression of cold resistance related genes is up-regulated.The expression of fmlhy,fmcbfl and fmcor413 are 57.47 times,19.2 times and 193.62 times higher than that in female parent control respectively;The expression levels of fmnac11 and fmnac12 genes were 210.1 times and 42.22 times higher than those of their maternal control.The differential expression of cold resistance related genes led to the cold resistance advantage of F1 generation.3.Cloning of fmnacll and its role in response to low temperature stressThe fmnacll overexpression vector was constructed and transformed into Benshi tobacco,and four transgenic positive lines were identified.The over expressed FmNAC11 tobacco plants were robust,thick branches and leaves,and showed obvious cold tolerance under low temperature stress.The content of MDA and relative conductivity of transgenic tobacco were lower than that of non transgenic control by 2.2%and 38.4%,the content of soluble protein and soluble sugar were higher than that of control tobacco by 34.1%and 65.5%,and the activities of antioxidant enzymes(SOD and POD)were higher than that of control tobacco by 275.7%and 3.6%,respectively;The expression levels of auxin synthase genes IAA and AUX1 were 164.8 times and 19.8 times that of non transgenic control respectively.Under cold stress,the expression levels of ICE,DREB and CBF1 genes related to cold resistance were 54.61 times,31.82 times and 10.94 times that of ordinary tobacco,and the expression levels of cat and SOD enzyme genes related to ROS clearance were 23.65 times and 6.04 times that of control respectively.Overexpression of fmnacll gene in tobacco can positively regulate the expression of defense related genes and increase the cold resistance of plants under low temperature stress. |
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