Screening And Analysis Of Flavonoid Biosynthesis Related Genes In Tengcha

Ampelopsis grossedentata(A.grossedentata)is one of the plants with the most abundant flavonoid content.Dihydromyricetin is then most important flavonoid component in A.grossedentata,which has antibacterial,hypoglycemic,hypolipidemic and antihypertensive properties.It has a variety of functions in dihydromyricetin,such as antibacterial,lowering blood sugar,lowering blood fat,anti-hypertension,anti-oxidation,liver protection,anti-tumor,etc.The content of dihydromyricetin in A.grossedentata leaves at different maturity levels varies greatly,close to 4 times.But the molecular mechanism is still unclear.In this paper,A.grossedentata leaves with different maturities were used as materials,the content of dihydromyricetin was detected by HPLC,the differentially expressed genes were analyzed by transcriptome sequencing technology,and the differential genes were detected in A.grossedentata leaves with different maturity by fluorescence quantitative PCR technology.The expression level of dihydromyricetin and the correlation between the content of dihydromyricetin and the expression of differential genes were verified to clarify the molecular mechanism of significant differences in dihydromyricetin in A.grossedentata leaves with different maturity.The main results are as follows:1.Using the HPLC method to determine the content of dihydromyricetin in A.grossedentata leaves(tender leaves,middle leaves,and old leaves),the test results show that the content of dihydromyricetin in the young leaves of A.grossedentata is as high as 42.03%,33.6% in the middle leaves,and 12.01% in the old leaves.The content of dihydromyricetin in the leaves of A.grossedentata at three maturity levels differed greatly,especially the young leaves and old leaves,it was nearly 4 times different.Therefore,the content of dihydromyricetin in A.grossedentata at different maturity stages gradually decreases with the increase of maturity,namely,young leaves>middle leaves>old leaves.2.Through transcriptome sequencing,a total of 306663600 Clean reads were obtained from A.grossedentata samples.The numbers of Unigenes successfully annotated in NR,NT,KO,Swissprot,PFAM,GO,and KOG databases are 81693,117488,30519,56889,55466,57640 and 20711 respectively.Through differential gene screening,6103 differential genes were obtained,of which 4350 differential genes were obtained in old leaves and young leaves,in that 1965 were up-regulated and 2,385 were down-regulated;a total of 1420 differential genes were obtained in middle and young leaves.Among them,475 were up-regulated and 945 were down-regulated.A total of 333 differential genes were obtained in the old and middle leaves,of which 120 were up-regulated and 213 were down-regulated.According to the results of differential gene enrichment,8 related genes in the flavonoid biosynthetic pathway were selected,namely PAL,C4 H,4CL,CHS,CHI,F3 H,F3′H,F3′5′H.3.Using fluorescence quantitative PCR technology to detect different maturity of A.grossedentata leaves,the expression levels of each target gene are different.On the whole,each target gene decreases with the increase of leaf maturity,in that,the expression levels of PAL,C4 H,4CL,F3 H,and F3’H in the three maturity levels of A.grossedentata were all decreased significantly;while the expression levels of CHS and F3’5’H were the highest in young leaves,and none in middle and old leaves.Besides,the gene CHI did not change significantly in young buds,middle leaves or old leaves.It can be inferred that PAL,C4 H,4CL,F3 H,F3’H are the key genes for the huge difference in the content of flavonoid biocomplexes in A.grossedentata leaves with different maturity,and CHS and F3′5′H are the key genes for the high content of flavonoid biocomplexes in the youngest leaves of A.grossedentata.4.The correlation analysis between the target gene and dihydromyricetin of different maturity levels of A.grossedentata showed that the expression of PAL,C4 H,4CL,CHS,CHI,F3 H,F3′5′H and F3′H is positively correlated with the content of dihydromyricetin.Among them,the correlation coefficients of PAL,C4 H,and 4CL are greater than 0.9,it means they are significantly related to the content of dihydromyricetin;the correlation coefficients of CHS,F3 H,and F3′5′H are above 0.75,and the content of dihydromyricetin is highly correlated;The correlation coefficient between CHI and F3′H is small,so CHI and F3′H have a little effect on the content of dihydromyricetin.Flavonoids have rich pharmacological activities.A.grossedentata is rich in flavonoids,among which dihydromyricetin is the main one.From a molecular perspective,it is helpful to study the reasons for the huge difference in flavonoid content in A.grossedentata with different maturity levels.Better development and utilization of A.grossedentata germplasm resources,while providing a theoretical basis and scientific basis for better research on flavonoids.