| Scutellaria baicalensis is a perennial herb belong to Lamiaceae family.With the functions of clearing heat,removing dampness,purging fire and detoxification,its dried root is used as a common medicinal material in China.In recent years,the wild resources of S.baicalensis have been exhausted,and the cultivar has been degraded.Extremly seasonal drought in some areas leave the active ingredients low and unstable,resulting in risks for clinical medication.However,moderate drought promotes the synthesis and accumulation of active ingredients in S.baicalensis.Therefore,it is necessary to explore transcription factors involved in the synthesis of active ingredients which response to drought stress,and elucidate the mechanism of drought stress regulating the biosynthesis of active ingredients at the molecular level.It will lay the foundation for improving the content,and cultivating new varieties of S.baicalensis with high-quality.Based on the previous transcriptome sequencing and data analysis,SbMYB transcription factors were screened.The potted annual and the cultivated tissue culture seedlings of S.baicalensis were used as experimental materials,and the drought stress was carried out through anhydrous treatment and the addition of different concentrations of PEG.The relative expression levels of seven key enzyme genes and four SbMYB transcription factor were detected by qRT-PCR,and HPLC was used to detect the content of active components in samples.By analyzing its dynamic changes and the correlation between them,the regulatory relationship among the active components,key enzyme genes and transcription factors was explored,and the molecular mechanism of SbMYB regulating the biosynthesis of active components was preliminarily revealed.The main results are as follows:1.Moderate drought stress promoted the accumulation of active ingredients in the roots,stems and leaves of S.baicalensis.The total content of the eight active ingredients in the roots and stems of S.baicalensis was the highest(217.95 mg/g;12.38 mg/g)under drought for 20 days,which was significantly increased 22.19%and 84.60%by compared with the control group(178.36 mg/g;6.62 mg/g);The six components including scutellarin in the roots reached their peaks on the 15th—20th day of drought,and increased significantly compared with the control group(P<0.01);The content of the seven active ingredients in the stems except scutellarein increased with the aggravation of the drought stress;the content of the active ingredients in the leaves all showed that the treatment group was higher than the control group during the drought.2.Both the SbMYB transcription factor and the key enzyme genes in the baicalin synthesis pathway responded significantly to drought stress.The relative expression levels of seven key enzyme genes and four transcription factors in the roots of S.baicalensis reached the highest on the 20th day of drought,which was extremely significantly higher than that of the control(P<0.01).2.The expression levels of PAL and other genes,MYB2,MYB11 transcription factor in stems and leaves were significantly up-regulated during drought stress(P<0.05).3.There is a significant correlation between the expression of SbMYB transcription factor and key enzyme genes in the baicalin synthesis pathway and the content of active ingredients under drought stress conditions.(1)The seven key enzyme genes in the roots of S.baicalensis have a very significant positive correlation with baicalin,wogonoside,baicalein and wogonin(P<0.01).The baicalin,scutellarin,wogonoside and chrysin of stems and leaves are extremely significantly correlated with key enzyme genes PAL,CHI,FNSII-2(P<0.01).(2)The SbMYB in the roots showed a very significant positive correlation with wogonoside,baicalein and wogonin(P<0.01),and it had a significant positive correlation with scutellarin(P<0.05)in stmes;MYB2 and MYB11 is positively correlated with the content of active ingredients in leaves(P<0.01).(3)The SbMYB in roots showed a very significant positive correlation with key enzyme genes except CHI(P<0.01),and there was also a certain correlation in stems and leaves.4.Optimized and established the tissue culture regeneration system of S.baicalensis.The results showed that the stem is the best explant for inducing callus of S.baicalensis.The best induction medium is:MS+1.0 mg/L 6-BA+0.5 mg/L NAA,and the induction period was about 20 days.The best medium for inducing callus differentiation is:MS+1.0 mg/L 6-BA+0.5 mg/L NAA.When MS medium without any hormone is used as the rooting medium,the roots of the tissue cultured seedlings develop best,suitable for transplanting,and the rooting rate is as high as 93.3%.The optimal light intensity for the growth of the tissue cultured seedlings is 8000 lx,the light time is 12 h/d,and the optimal growth temperature is 22?.5.The results of PEG drought stress showed that the expression of SbMYB transcription factors and key enzyme genes had different effects on the response of PEG concentration and the accumulation of active ingredients.High-concentration(2.5%?10.0%)PEG stress treatment inhibited the total content of the six active ingredients in the tissue cultured seedlings of S.baicalensis,while the low-concentration group(0.5%?2.0%)showed promoting effects.The content of five components and the expression of six genes were extremely significantly increased under short-term(3 days)low-concentration(0.5%?1.5%)PEG stress(P<0.01),but the expression of F8H was down-regulated.Moderate(6 days;0.5%?1.5%)PEG stress promoted the expression of MYB2 and MYB11,while MYB7 and MYB15 were up-regulated under short-term(3 days)high-concentration(2.5%?10.0%)stress.Correlation analysis showed that the seven key enzyme genes in the tissue cultured seedlings showed a very significant correlation with scutellarin,scutellarein,baicalin and wogonoside(P<0.01);MYB2 and MYB11 are significantly positively correlated with scutellarin and baicalin respectively(P<0.01),while MYB7 and MYB15 are negatively correlated with them;four SbMYB were significantly correlated with seven key enzyme genes(P<0.01).The above research results indicate that the SbMYB transcription factor may promote the biosynthesis of active components in regulating the expression of key enzyme genes in the baicalin synthesis pathway by response to drought stress,and then enhancing its own drought resistance ability.It will provide new ideas and theoretical basis for cultivating high-quality new varieties through genetic engineering,germplasm innovation and hybrid breeding,and for solve the problems of germplasm degradation,low medicinal active ingredients and unstable quality of Chinese medicinal materials. |
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