| Leaves of ginkgo are rich in flavonoids,but with the increase of seedling age,the content of flavonoids in leaves gradually decreases.In production practices,truncated treatment are generally adopted to improve the content of flavonoids in leaves of ginkgo.R2R3-MYB transcription factor,as one of the largest family in the MYB gene family,was also involved in the regulation of ginkgo flavonoid metabolism.Therefore,it is of great significance to study the effect of truncation on the flavonoid metabolism pathway in leaves of ginkgo and to explore the differential expression of R2R3-MYB transcription factor related to flavonoid metabolism by truncating.In this study,the contents of total flavonoid glycosides,quercetin,kaempferol and Isorhamnetin in leaves of ginkgo during the growth cycle were studied by cutting off the grafted seedlings,as well as the changes of the deoxidizing ability of flavonoids and the free radical scavenging rate.In order to measure the gene expression more accurately,on the basis of screening the best reference genes suitable for this study,the expression levels of 9 key enzyme structure genes（GbPAL,GbC4H,Gb4CL,GbCHS,GbF3H,GbFLS,GbDFR,GbANS,GbUFGT）in the flavonoid metabolism pathway of ginkgo were further studied under the truncated treatment.The identification and analysis of the R2R3-MYB gene family was carried out based on the ginkgo genomic information.The RNA-Seq technology was used to screen the R2R3-MYB gene related to flavonoids after truncation.Two potential GbR2R3-MYB genes（Gb＿22884,Gb＿39852）were obtained and genetic transformation were conducted.The main findings are as follows:1.During the one-year growth cycle,the total flavonoid glycoside content of leaves showed a gradual downward trend.At 15 days after germination,the total flavonoid glycoside content of leaves reached the peak(21.84 mg·g-1)The total flavonoid glycoside content was the lowest at165 days after germination(4.92 mg·g-1).In general,the content of total flavonoid glycosides,quercetin,kaempferol and Isorhamnetin in leaves of ginkgo,all showed a trend of gradual decrease in the annual growth cycle after the treatment.Compared with the control group,the content of total flavonoids glycosides in leaves of ginkgo in the treatment group increased by at least 5.92%.At 15 days after germination,the content of quercetin in leaves of ginkgo in the treatment group increased by 9.12%compared with that in the control group（P<0.05）,The contents of kaempferol in the treatment group were significantly higher than those in the control group（P<0.05）.There was no significant difference in the content of Isorhamnetin between the treatment group and the control group（P>0.05）.2.In the annual growth cycle after truncated treatment,the deoxidizing ability of leaves was significantly higher than that of the control group at 15,45,75 and 135 days after bud（P<0.05）.After the treatment,the deoxidizing ability was the highest at 15 days after bud（0.72）,which was4.35%higher than that of the control group.During the one-year growth cycle after the truncated treatment,the free radical scavenging rate of leaves in ginkgo biloba changed significantly during the first three measurement periods（P<0.05）,but there was no significant change trend during the last three measurement periods（P>0.05）.3.The stability of 7 candidate internal reference was analyzed in different tissues of ginkgo and in different development stages of leaves of ginkgo,the results showed that GbUBQ was the most stable gene expressed in the leaves of male plants at different development stages of ginkgo biloba,so GbUBQ was selected as the internal reference gene in this study.The expression levels of four genes（GbPAL,GbC4H,Gb4CL,GbCHS）in the upstream of the flavonoid metabolic pathway of ginkgo biloba were significantly increased compared with those in the control group.The differences in expression levels of these four genes between the treatment group and the control group also gradually narrowed.In the leaves of ginkgo biloba at six different development stages,the expression trend of the above four genes was positively correlated with the change trend of flavonoid content.The expression levels of the three genes（GbF3H,GbFLS,GbDFR）in the middle of the flavonoid metabolic pathway decreased gradually during an annual growth cycle.The expression levels of GbANS and GbUFGT in the downstream of the flavonoid metabolic pathway were different from the expression trends of the remaining genes,and showed a trend of first increasing and then decreasing in a growth cycle.The expression level of GbANS reached its peak at 45 days after bud,and the expression level of GbUFGT reached its peak at 75days after bud.4.A total of 69 GbR2R3-MYB genes were screened from the ginkgo genome database.These regulatory genes were randomly distributed on the 12 chromosomes of ginkgo.Analysis of the protein conserved motifs of 69 GbR2R3-MYB genes revealed that there were 20 conserved motifs in these genes,all of them contained motif 1～3.The relative positions of the conserved motifs on different genes are consistent.69 GbR2R3-MYB genes were classified into 6 MYB subfamilies,and intron exon analysis was performed.About 78.26%of the total genes contained 2 introns and3 exons.There were significant differences in the expression of 69 Gb R2R3-MYB genes in different tissues.5.After the truncated treatment,there were 5418 differentially expressed genes in leaves of ginkgo,of which 2620 genes were significantly up-regulated and 2798 genes were significantly down-regulated（P<0.05）.16 GbR2R3-MYB genes were differentially expressed under the treatment of truncation,among which 11 GbR2R3-MYB genes were significantly increased（P<0.05）.In combination with the differential expression of these genes in leaves at different development stages,Gb＿22884 and Gb＿39852 genes were selected for cloning,and the over-expression vectors were successfully constructed and transformed into arabidopsis for verification. |
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