Physiological And Ecological Changes Of Scutellaria Baicalensis Georgi Under Drought Stress And Mlecular Ecological Mechanism Of Baicalin Biosynthesis

Scutellaria baicalensis Georgi is a perennial herb of the family Lamiaceae.It is mainly distributed in Northeast China,North China,Northwest China and other places.The different ecological factors lead to quality difference among Radix Scutellariae,which seriously affected the stability of raw materials and product quality from S.baicalensis.My research group has confirmed that water was the main restrictive ecological factor affecting the quality of S.baicalensis,and it was found that moderate drought stress could promote the accumulation of flavonoids.Therefore,based on the previous studies and from the perspective of plant physiology and molecular ecology,the research on the response of the main active components of S.baicalensis to drought stress was carried out systematically.To explore the molecular ecological mechanism of the quality formation of S S.baicalensis has important theoretical significance and application value in perfecting scientific cultivation technology of S.baicalensis and realizing quality control of S.baicalensis.The study first explored the effects of long-term drought stress on secondary metabolism of S.baicalensis,and tried to find out the genetic characteristics of secondary metabolism accumulation of S.baicalensis and the common effects of drought stress.On this basis,the short-term progressive drought stress and the short-term drought stress re-water study were carried out during the key periods of the accumulation of the main active components of S.baicalensis,to investigate the controlled model of the accumulation of active constituents of S.baicalensis in response to drought stress,and to study the transcriptional regulation mechanism of S.baicalensis in response to drought stress by means of high throughput.Combined with the researches on photosynthetic physiology and resistance physiology,the study of resistance physiology revealed the molecular physiological and ecological mechanism of the accumulation of active components of S.baicalensis in response to drought stress,aiming to provide scientific basis and technical support for promoting the quality and ecological regulation of Radix Scutellariae.The main findings are as follows:1.Design expert 8.05 b software Box-Behnken method was used to optimize the microwave assisted extraction of S.baicalensis active ingredients under single factor.The best extraction conditions were obtained: extraction time was 3.3 min,extraction temperature was 79.40 ?,liquid material ratio was 32.60 mL/g,and ethanol concentration was 66.30%.Under these conditions,the yield of flavonoids in S.baicalensis was 19.79%,and the difference was less than that of software prediction.At the same time,the yield was higher than that by traditional heat reflux extraction method,which had the characteristics of short time and high efficiency,providing a good method for the extraction of flavonoids in medicinal plants.It also lays a foundation for the study of synthesis mechanism of secondary metabolites of S.baicalensis.2.The result for long-term drought stress of annual or biennial potted S.baicalensis by simulated rainfall shown that Long-term severe drought stress significantly inhibited the growth of root,while mild drought stress had little effect on it.It was also found that long-term drought stress had little effect on baicalin content,but could significantly increase baicalein content.There were double peaks of baicalin content in June and September,and baicalin had obvious response to drought stress in this period,which may be the key period of water regulation of secondary metabolites of S.baicalensis.In addition,baicalin response to drought stress had a short-term effect,which may be related to be the secondary metabolic compensation mechanism of baicalin in response to drought.3.Biennial S.baicalensis was used as research material,and in water sensitive periods of baicalin accumulation,that was,the vegetative period(June)and the reproductive period(September),short-term progressive drought stress and short-term progressive drought stress rewatering of S.baicalensis were stduied.The results showed that baicalin content was significantly regulated by drought stress.Baicalin accumulated significantly during mild and moderate drought stress,and baicalin synthesis was inhibited by severe drought stress.After rewatering,the inhibition of baicalin biosynthesis was decreased.However,drought stress severely inhibited the growth of root in vegetative period,but little affected on root in the reproductive period.Therefore,reproductive growth period can be used as the potential water regulation of S.baicalensis.4.The correlation analysis of photosynthetic physiology,resistance physiology and the main active components of S.baicalensis showed that Pn,Gs and Tr of S.baicalensis were less affected in the early stage of drought stress,and the effects were increased with the extension of drought stress time.The actual photochemical efficiency(?PSII)decreased,the photosynthesis weakened and MDA began to accumulate,cell membrane was damaged,the activities of SOD,POD and CAT in the antioxidant system of S.baicalensis were increased,and the contents of Pro,SS and SP increased.The contents of baicalin and baicalein increased significantly.But under severe drought stress,Pn,Gs and Tr were inhibited significantly.At the same time,NPQ significantly increased,the original photosynthetic structure was damaged,MDA content continued to rise,SOD,POD and CAT activities were obviously inhibited,and SP content of osmotic adjustment materials decreased.At this time,the content of baicalin decreased,while the content of baicalein increased.It showed that the metabolism of baicalin and baicalin was compensated for the response of drought stress.5.The key enzymes in the metabolic synthesis pathway of baicalin under drought stress were studied.The results showed that the four key enzymes play an important role in the synthesis of baicalin.Compared with baicalein,baicalin accumulation was significantly affected by the key enzymes in the synthesis pathway.Short-term and moderate drought stress could enhance the activities of PAL,C4 H,4CL and CHS,and promoted the accumulation of baicalin.Long-term and severe drought stress inhibited the activities of 4 enzymes,and baicalin synthesis was inhibited.PAL and CHS had obvious effect on the regulation of baicalin in long-term drought stress.And C4 H,CHS and PAL had obvious effect on the regulation of baicalin in vegetative stage and reproductive stage respectively.6.SbPAL,sbC4 H,sb4CL,sbCHS,sbCHI,sbFNS,sbUBGAT and sbGUS genes of baicalin biosynthesis pathway were constructed by using 18 SrRNA as internal reference gene.The results showed that drought stress significantly affected the gene expression of flavonoid biosynthesis key enzymes in the root of S.baicalensis.In general,short-term drought stress contributed to the expression of the above genes.Severe drought stress inhibited the expression of key enzyme genes,but had different effects on different genes.Among them,the content of baicalin was significantly correlated with the expression of sbFNS under long-term drought stress.The content of baicalin in long-term drought stress was significantly correlated with SbFNS expression.The expression patterns of sbCHS and sbPAL were similar to the accumulation pattern of active components of S.baicalensis in biennial reproductive period,and were significantly changed by drought stress,sensitive to water response,and played an important role in the process of quality formation of S.baicalensis,which could be regarded as a candidate genes for quality control of S.baicalensis.7.Illmina 4000 sequencing platform was used to analyze the transcription sequence of Scutellaria baicalensis root in four different stages of drought stress,i.e.control,mild drought stress,moderate drought stress and severe drought stress in the reproductive period of drought stress.93.59 GB data were obtained and 57307 unigenes were spliced and annotated.A large number of differential expression genes were enriched in carbon metabolism,plant hormone signal transduction and phenylpropane biosynthesis including a large number of reactive oxygen scavenging genes,osmotic regulating substances synthesis genes.It was also found that a large number of transcriptional regulator genes were involved in the drought stress process,among which MYB(c49842.graph_c0)and WRKY(c59576.graph_c2)had high expression,which may play an important role in regulating the accumulation of baicalin under drought stress.In conclusion,short-term drought stress can be used as an effective measure to improve the quality of S.baicalensis and provide technical reference for standardized cultivation.However,the biosynthesis and accumulation of baicalin is not determined by a single factor,but is the result of multiple factors,such as photosynthesis,hormone,enzyme activity and gene expression.The following study will focus on the relationship between signal transduction,transcriptional regulation and the secondary metabolic pathways of baicalin under drought stress,and try to elucidate the regulation mechanism of transcriptional regulation factor on key enzyme gene expression.