Analysis Of Key Enzyme Activities And Gene Expression Of Flavonoid Biosynthetic Pathway In Chinese Fir Sterile Seed

Chinses fir[Cunninghamia lanceolata(Hook.)Lamb.]is one of the main afforestation tree species in the subtropical regions of southern China.It has a wide distribution area and a long history of cultivation.In the development of Chinese fir seed,abortion phenomenon is very serious,which seriously affects the quality of the seed.Previous studies have found that there are a lot of phenolic substances such as tannins in the Sterile seeds,and polyphenols such as tannins are the downstream metabolites of the flavonoid biosynthesis pathway through two families(low astringent seed rate family The rate of astringent seeds in L years is 24%,and the rate of astringent seeds in H series seeds is as high as 90%for consecutive years).The transcript level of early embryo development(20d after fertilization)was found to have multiple Unigenes in the flavonoid biosynthetic pathway.There was a significant difference in expression.this research mainly focused on the seeds of Chinese fir in 70d-100d(fertilization stage,proembryonic development stage,and multi-embryo selection stage)after pollination in L and H families.Liquid chromatograph was used to detect the metabolites related to the flavonoid biosynthetic pathway:quercetin,kaempferol,myricetin,catechin,and The content of catechins was determined by enzyme linked immunosorbent assay(ELISA),and the activities of CHS,CHI,F3’H,DFR,FLS,ANS,LAR and ANR were determined and their rates of different types of astringent seeds were compared.The activity of families and different developmental stages was studied.The expression of CHS,CHI,F3’H,DFR,FLS,ANS,LAR and ANR genes was studied by qPCR double standard curve method.The synthesis of these key enzymes was examined from the expression level.situation.In order to verify the performance of flavonoids in the formation of astringent seeds,it is necessary to analyze the dynamic expression changes of key enzymes and their genes in flavonoid biosynthesis pathways during the formation of astringent seeds.Preliminary research basis.The main research conclusions are as follows:(1)Through the study of the flavonoid-related metabolites in the three stages of embryonic fertilization,embryo development,and multi-embryo selection,the total flavonoid content in the Chinese fir seed development continued to increase.Lower than the low-astringent seed rate family,indicating that there may be less total flavonoids in the pine needles;flavonoid metabolic pathway metabolites quercetin,kaempferol,myricetin content decreased first and then rose,H family fir The contents of quercetin,kaempferol and myricetin in seeds were all lower than those in L family,indicating that there were fewer quercetin,kaempferol and myricetin in the pine needles of Cunninghamia lanceolata;the two family catechins in the downstream showed an upward trend.There was no significant difference between the familial and L-family.The epigallocatechin content first rose and then decreased.The content of catechins in the H-family Cunninghamia lanceolata seeds was higher than that in the L-family,indicating that the C.olitorifolia may contain more seeds.In tea,the content of anthocyanin decreased first and then rose.The content of anthocyanin in seed of H-family Cunninghamia lanceolata was lower than that in L-family,indicating that there were less anthocyanins in Cunninghamia lanceolata seeds.(2)It can be seen from the rule of enzyme activity that the activities of CHS and CHI enzymes in the upper flavonoid biosynthetic pathway during the seed development of Cunninghamia lanceolata are relatively high,showing an upward trend.The H family CHS enzyme activity is slightly lower than the L family but not much different The results showed that the abortion of Chinese fir has little effect on the activity of CHS and CHI enzymes.(3)The activity of F3’H enzyme in the middle reaches was high,and it showed an upward trend along with time.After 90 days,the activity of F3’H enzyme in the seeds of Healthy and astringent seeds began to show a large difference;the activity of DFR enzyme was higher,showing an upward trend.The DFR enzyme activity of the familial line was slightly lower than that of the L family line,indicating that the abortion of Chinese fir had little effect on the DFR enzyme activity;the activity of the FLS enzyme was lower,and it showed an upward trend as a whole over the time,showing significant differences since 70 days.(4)The activity of LAR enzyme in the lower reaches was also lower.The activity of the L family enzyme first decreased and then increased.The H family showed a decreasing trend.The activity of the two families was significantly different after 90 days.The activities of ANS and ANR were all higher.The activity of ANS enzyme increased first and then decreased after 70d-90d.Subsequently,the activity of ANS enzyme in H family continued to decline,while the activity of L family enzyme increased,and the ANR enzyme activity had a large difference at 70 days.The H family enzyme activity was significantly higher than that of the L family,but the H family showed a decreasing trend and the L family showed an upward trend.At the 100th day,the enzyme activity was equal.(5)The relative expression of the gene relative to the copy number can be seen that the upstream CHS,CHI gene expression levels are high,with the time up-regulated expression,compared to L group,H group CHS,CHI gene expression did not appear more Big change.(6)The F3’H and FLS gene expression levels in the middle reaches are high,and the DFR gene expression levels are low.In addition to the FLS gene,the F3 ’H and DFR genes are upregulated over time,compared to the L family,H family F3 ’H gene expression increased significantly,DFR gene expression decreased,FLS gene expression decreased significantly.(7)LAR and ANR gene expression levels were low,and LAR,ANR and other genes all showed an upward trend with time.The expression patterns of LAR and ANR genes in the two homes began to show obvious differences after 80 days.The LAR gene of H family showed a large up-regulated expression trend,while ANR showed a down-regulated expression.