Effect Of Frost And The Underlyning Molecular Mechanism On The Accumulation Of Flavonoids In Mulberry Leaves

Mulberry leaves are the dry leaves of Morus alba L.(family Moraceae),which are one of the medicinal and food dual-purpose varieties and also are commonly used traditional Chinese medicines for evacuating wind-heat,clearing away lung-heat and relieving dryness as well as removing liver-fire for improving eyesight.The Pharmacopoeia of the People's Republic of China describes that mulberry leaves are harvested after the first frost.Many medicinal herbs also records that frosting mulberry leaves are superior in quality and medicinal properties,but the molecular mechanism of changes in the chemical composition of mulberry leaves and their effects on the change of chemical substances after frost is still not fully elucidated.In this thesis,High Performance Liquid Chromatography and Mass spectrometry was used to study the dynamic changes of the main secondary metabolites of mulberry leaves,and molecular biology techniques and methods were used to investigate the gene expression level of key enzymes in flavonoids biosynthesis pathway of mulberry leaves and the key gene cloning and function in the final step of the biosynthetic pathway.It will reveal the internal molecular mechanism that form high-quality mulberry leaves after frost,providing scientific basis for the rationality of mulberry leaves harvested by frost.The specific research contents are as follows: 1.Study on the dynamic changes of flavonoids in mulberry leaves before and after frost. High Performance Liquid Chromatography(HPLC)-Mass spectrometry(MS)was used for the qualitative analysis of flavonoids in mulberry leaves.Ten compounds were identified,mainly flavonol glycosides.A method for the simultaneous determination of six major flavonoids(chlorogenic acid,rutin,isoquercitrin,acetylisoquercitrin,astragalin,acetylastrgalin)in mulberry leaves was established,and the dynamic changes of those six compounds from May to November were studied for the first time.Quantitative analysis showed that the contents of six flavonoids were higher at lower temperature in May,began to fall with increasing tempetature in June,lowest at higher temperature in August,and gradually increased when the temperature became lower in September.The maximum contents of flavonol glycosides reached after the frost-thropping(October 23)until November.This conclusion verified the rationality of harvesting after the first frost of mulberry leaves.SPSS software was used to analyze the correlation between climate temperature and the contents of flaovoids in mulberry leaves of the different growing seasons.The results showed that climate temperature had a great influence on the accumulation of flavonoids in mulberry leaves.When temperature was higher,the contents of flavonoids decreased,and when temperature was lower,the contents of flavonoids increased,suggesting that the lower temperature may be conducive to the accumulation of flavonoids.2.Effects of frost on the expression level of key enzymes in the flavonoids synthetic pathway in mulberry leaves.The flavonoid biosynthetic pathway is a phenylpropanoid pathway that begins with phenylalanine and requires the participation of several key enzymes.Phenylalanine ammonia-lyase(PAL)is the first key enzyme involved in the formation of flavonoids.4-Coumarate?Co A ligase(4CL)acts on the final step of the phenylpropanoid pathway and is a key enzyme in the formation of flavonoids and lignin substances.Chalcone synthase(CHS)is a key enzyme in the flavonoid pathway.UDP glucose flavonoid-3-O-glycosyltranferase(UFGT)is the last key enzyme in the flavonol glycoside synthesis pathway.In this dissertation,the four enzyme genes of flavonoids biosynthetic pathway were selected to study their expression levels in mulberry leaves in different growing seasons,and their correlation with accumulation of flavonol glycosides components in mulberry leaves in different growing seasons was analyzed:(1)The PAL,4CL,CHS and UFGT genes of mulberry leaves were cloned and sequenced to obtain four gene sequences.The homology analysis was performed in the Gen Bank database to verify that these four genes were expressed in mulberry leaves.(2)Expression levels of PAL,4CL,CHS and UFGT in mulberry leaves in different growing seasons were studied by q RT-PCR for the first time.It was found that the expression levels of these four genes were significantly different in different growth seasons of mulberry leaves.At the end of August and early September,the expression levels of the four genes were lower,and after Frost‘s Descent,they reached a higher value in November.Experimental data showed that the maximum expression level of PAL gene was 0.2083,which was 42.5 times of the lowest expression level;the maximum expression level of 4CL gene was 0.6262,which was 61.4 times of the lowest expression level;the maximum expression level of CHS gene was 0.2093,which was 299 times of the lowest expression level The maximum expression level of UFGT gene was 0.1116,which was 12.4 times of the lowest expression level.(3)The correlation between the expression levels of PAL,4CL,CHS,and UFGT genes and the accumulation of flavonol glycosides in different growing seasons was analyzed by SPSS software.There was a certain degree of positive correlation between gene expression level of the four enzymes and contents of isoquerctrin,acetylisoquercitrin,astragalin and acetylastragalin,and the correlation with rutin content was small.Especially the expression levels of 4CL,CHS and UFGT genes were significantly and positive correlated with isoquercitrin and astragalin content(P<0.01).The correlations between temperature and expression levels of the four genes were also analyzed.Temperature was negatively correlated with the expression of PAL and 4CL genes(P<0.05),and significantly and negative correlated with the expression of CHS and UFGT genes(P<0.01).The results suggest that low temperature may induce the expression level of these enzyme genes.3.Gene cloning,prokaryotic expression and functional analysis of flavonoid-3-O-glucosyltransferase(Ma UFGT)of mulberry leaves.Mulberry flavonoid-3-O-glucanotransferase gene was cloned by RT-PCR(reverse transcription PCR)using the c DNA of young leaves of mulberry as a template and named Ma UFGT(Gen Bank accession number MH198038).Its open reading frame(ORF)was 1455 bp long and encoded 484 amino acids.The predicted molecular weight of the protein was 54.491 KD and the isoelectric point was 5.3.The protein sequence contains a GTB topology,confirming that Ma UFGT belongs to the GTB type family of glycosyltransferases.The full-length Ma UFGT gene was synthesized based on designed spliced primers by codon-optimized whole gene synthetic method.The Ma UFGT gene was cloned into the E.coli expression vector p Czn1,and the recombinant plasmid p Czn1-Ma UFGT was constructed.The target band was successfully obtained by IPTG induction.The expressed protein is approximately 54 KD in size and is mainly present in the form of inclusion bodies.After the recombinant protein was denatured,renatured and purified,in vitro enzymatic activity was determined by HPLC.The Ma UFGT protein obtained in this experiment was found to transfer UDP-glucose glycosyl moieties to the 3-hydroxyl group of quercetin and kaempferol to form the corresponding isoquercitrin and astragalin in vitro.It verified that the enzyme had the function of a glycosyl transferase.4.Temperature on Gene Expression of Key Enzymes in the Pathogenesis of Flavonoids Synthesis and accumulation of flavonoids in Mulberry Leaves.?Frost? here is the temperature change.The accumulation of flavonoids and the expression of PAL,4CL,CHS,and UFGT after frost increased significantly.In order to verify the effect of temperature on the expression of key enzymes in the synthetic pathway of flavonoids,artificial temperature chambers were used to simulate the artificial climate settings in August(35°C/30°C)and before and after the frostfall(15°C/10°C).Mulberry seedlings were cultivated,and transcriptome sequencing was performed on mulberry leaf samples under two temperature conditions.As a result,raw data with a total number exceeding 12 G were obtained,and the percentage of Q30 bases was 94.58%.Sequences of clean reads of each sample were compared with the designated genome of Morus notabilis(reference genome).Efficiency of the comparison was 70.92% or more.Analysis of differentially expressed genes revealed that the total DEGs were 1490,with 755 up regulated and 735 down regulated.There were 15 DEGs related to the biosynthesis of flavonoids,including 2 PAL genes,2 C4 H genes,1 4CL gene,8 CHS genes and 2 shikimate hydroxycinnamoyl transferase genes.The PAL,C4 H,4CL,and CHS genes were significantly up-regulated.HPLC analysis showed that the peak areas of chlorogenic acid,rutin,isoquercitrin and astragalin in mulberry leaves increased under low temperature conditions compared with high temperatures.The experimental results confirmed that low temperature can induce the expression of key enzyme genes in the flavonoid synthesis pathway of mulberry,which is beneficial to the accumulation of flavonoids.