| Fraxinus mandshurica is one of the fine tree species in Northeast my country and has a very high value.In this study,the new hybrid progeny cultivar"Dongshui 1601"of Fraxinus sogdiana and its female parent M8 clone were used as research materials to determine and analyze the main phenotypic differences between parents and offspring.Group analysis analyzed the differential formation mechanism of parental and progeny leaves,and performed bioinformatics and expression pattern analysis on the growth-regulating factors（GRFs）transcription factor family that played a key role in leaf development.The FmGRF1 gene was successfully cloned and 35S:FmGRF1 was constructed.The overexpression of FmGRF1 in ash plants laid a solid foundation for the improvement of ash germplasm resources and the analysis of gene functions.The main findings are as follows:1.Analysis of the main phenotypic differences between the new interspecific hybrid varieties and their parents of Fraxinus mandshuricaThe main phenotypic data of xylem and leaves of the new interspecific hybrid progeny variety"Dongshui 1601"and the female parent M8 clones（referred to as 1601 and M8,respectively）were determined,and differences were analyzed.In terms of xylem development,the xylem radial development cycle of 1601 is 5.14-8.21,and the xylem radial development cycle of M8 is 5.14-8.8.The xylem radial development cycle of 1601 is 2 weeks longer than that of its parent M8 in one year;1601 latewood units The number of area cells is 2000±216mm-2,which is 83.33%of M8,indicating that 1601 cells are larger than M8,and there is a significant difference;1601 lignin content is 22.89±0.93%,which is 86.65%of M8 content;and 1601 fibers The element content was 49.12±2.83%,which was 1.14 times that of M8 and significantly higher than that of M8.In terms of leaf phenotype,the leaflet length of 1601 is38.84±3.68 mm,which is 58.02%of the M8 leaflet;the leaflet width is 26.28±2.29 mm,which is 86.48%of the M8 leaflet;the leaflet area is 713.33±143.10 mm2,which is 53.78%of the M8leaflet;the net photosynthetic rate was 9.80±1.87μmol CO2m-2s-1,which was 1.43 times that of M8 leaflet;M8 leaves were consistently higher than 1601 leaves in terms of water loss rate in isolated leaves,with significant differences from 5 h to 24 h.2.Transcriptome analysis of leaves of new interspecific hybrids and female parent of Fraxinus mandshurica1601 and M8 ash leaf RNA was extracted for transcriptome analysis.The results showed that there were 11,321 differentially expressed genes in leaves,of which 6,008 genes were up-regulated and 5,313 genes were down-regulated in 1601 leaves;KEGG enrichment analysis showed that more than 20 pathways were significantly enriched in parental leaves（P<0.05）,15 pathways were extremely significantly enriched（P<0.01）,including flavonoid biosynthesis,isoflavone biosynthesis,anthocyanin biosynthesis,and flavonoid and flavonol biosynthesis pathways.The differentially expressed genes in the flavonoid biosynthesis pathway and the phytohormone signal transduction pathway were drawn heat map to analyze the regulatory mechanism of parent-child phenotype differences.Among them,1 BZ1,1 CHS and 1 CHS as key genes in the flavonoid biosynthesis pathway and 2 CYP73As were highly expressed in1601 leaves,the expression level of BZ1 in 1601 leaves was 1.57 times that of M8,the expression level of CHS in 1601 leaves was 7.41 times that of M8,and the expression levels of the two CYP73As were 2.45 times and 2.64 times,respectively.Among the 8 AUX/IAA genes that play an important role in the auxin signal transduction pathway,6 genes are highly expressed in 1601,which are 3.74,1.51,1.39,1.47,86.76 and 1.41 times the expression in M8,respectively.;2 genes were lowly expressed in 1601,and the expression in M8 was 34.87%and 52.60%,respectively;5 BZR1/2 genes,as key genes in the BRs synthesis pathway,were differentially expressed between 1601 and M8.One of the genes was highly expressed in 1601,and the expression level was 1.64 times that of M8;the remaining four genes were lowly expressed in 1601,and the expression levels were 62.68%,76.19%,51.65%,and 32.37%of M8,respectively.Through differentially expressed gene analysis,it was found that GRF and TCP transcription factor families had a large number of differentially expressed genes in parental leaves,including 4 GRF and 10 TCP transcription factors.Two GRF genes were highly expressed in 1601 leaves,and the expression levels were 2.71 times and 1.83 times that of M8;two GRF genes were expressed at lower levels in 1601 leaves,which were 28.00%and87.87%of M8,respectively.The expression levels of 5 TCP genes were significantly higher in1601 leaves than in M8,and the expression levels were 1.29,2.29,1.82,1.37 and 1.63 times,respectively;the expression levels of 5 TCP genes in 1601 leaves were significantly lower than those in M8,The amount is 73.10%,55.21%,54.20%,50.97%and 41.02%of M8,respectively.3.Cloning and expression pattern analysis of FmGRF gene from Fraxinus mandshuricaThe phylogenetic tree of the FmGRF gene family was constructed and the structural domain was analyzed,and the expression patterns of all FmGRFs in different parts of ash were analyzed.There are members expressed in the xylem,phloem and leaves of ash,and participate in the regulation of the development of the xylem,phloem and leaves of ash.The Fraxinus mandshurica FMGRF1 gene was cloned,and the full length of the coding region was 1953 bp,encoding 651 amino acids.The 35S:FmGRF1-overexpressing ash plants were constructed and verified by q RT-PCR.It was determined that the leaflet length of 35S:FmGRF1-overexpressing ash plants at 60 days was 10.46±1.76 mm,which was 1.56 times that of WT tissue culture seedlings of the same age.Significantly higher than that of WT tissue culture seedlings of the same age;at the same time,q RT-PCR was performed to analyze the expression of four genes encoding DELLA proteins that may interact with GRF transcription factors.The expression levels were 40.86%,50.18%,35.17%and 49.44%of WT respectively,all of which were significantly down-regulated. |
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