| Cajanus cajan(L.)Millsp is the fifth most important legume crop mainly cultivated in semitropical and tropical regions of the world.The main secondary metabolites in C.cajan are phenols,among which cajaninstilbene acid is the most representative active compound.Phenolic compounds are known as the primary phytochemicals responsible for the health benefits of C.cajan.However,there are fluctuations in the content of phenolic compounds caused by factors such as season,variety,climate and geography,which directly affect the application of pigeon pea extract in the nutrition/pharmaceutical industry.Plant tissue culture technology is considered to be one of the most promising alternative methods due to its outstanding advantages such as being unaffected by geographical,climatic and other factors and capable of stably producing plant secondary metabolic active components.Among them,hairy root culture(CCHRCS)has the advantages of safety,reliability,genetic stability,rapid growth,high synthesis ability of secondary metabolites,easy preservation and large-scale production has become an ideal biological system for the efficient production of secondary metabolic active components of medicinal plants and the study of their biosynthesis regulation mechanisms.In this study,a UPLC-Qq Q-MS/MS method for the qualitative and quantitative analysis of phenols was initially established.The developed UPLC-Qq Q-MS/MS method was used to guide the establishment of CCHRCS with the high productivity of phenols,which could act as a model platform for studying their biosynthesis mechanisms.Subsequently,CCHRCS were treated by UV-B radiation,and the target active ingredients accumulation was used as an indicator to optimize the optimal operational time of UV-B radiation.Eventually,the potential key enzyme genes regulating the biosynthesis of phenolic active components in CCHRCS under UV-B stress were preliminarily identified by analyzing the variation of phenolic active components content before and after UV-B stress treatment,combined with the expression difference of candidate enzyme genes for biosynthesis of phenolic active components.The main research contents and results are summarized as follows:1.Development of UPLC-MS/MS method for the determination of principal active phenolics in C.cajan,conditions for the determination of 11 phenolics:Chromatographic conditions:ZORBAX Eclipse Plus C18 column(50 mm×2.1 mm inner diameter,1.8μm);mobile phase consisted of acetonitrile(A)and water(B);gradient elution was 0-2 min,25-35%A,2-3.5min,35-90%A,3.5-5 min,90%A,5-5.1 min,90-25%A,5.1-6min,25%A;column temperature was 30℃,flow rate was 0.4 m L/min,injection volume was1μL.MS conditions:Apart from pinostrobin and cajaninstilbene acid,the rest nine analytes exhibited better responses in the negative ionization mode.Under the optimal MRM conditions,the target compound ion pairs are orientin m/z 447.1,327.0(ESI~-),isovitexin m/z 431.1,341.0(ESI~-),vitexin m/z 431.1,311.0(ESI~-),genistin m/z 431.1,268.1(ESI~-),luteolin m/z 284.7,133.1(ESI~-),genistein m/z 269.0,133.1(ESI~-),apigenin m/z 269.0,117.1(ESI~-),isorhamnetin m/z 315.1,300.0(ESI~-),biochanin A m/z 283.1,268.1(ESI~-),pinostrobin m/z 271.1,167.1(ESI~+),cajaninstilbene acid m/z 339.0,283.2(ESI~+).The proposed UPLC-Qq Q-MS/MS method,in the very short run time(6 min),not only could effectively eliminate the interference of other non-target impurities but also could quail-quantitatively determine 11 target ingredients in CCHRCS quickly,sensitively and accurately.So the developed UPLC-Qq Q-MS/MS methods were suitable for the accurate analysis of trace ingredients in CCHRCS.2.Establishment of CCHRCS system for the efficient production of phenolic.(1)An optimal transformation rate(70.92±8.14%)was obtained when 2-week-old C.cajan leaf explants were co-cultured with A.rhizogenes LBA9402 in the medium containing 75μM acetosyringone and 400 mg/L cefotaxime sodium for 2 days in the 10-day induction cycle.Based on the biomass production and the accumulation of active ingredients,the high-productive phenolic root line PPHRL II was successfully selected from 12 candidate root lines and confirmed by PCR amplification.(2)Systematically optimize the liquid culture conditions and inoculation amount of CCHRCS PPHRL II.The optimal culture conditions were as follows:1/2 MS liquid culture medium,initial p H 5.8,sucrose concentration 3.0%,2.0%inoculation amount,temperature27℃and growth period 42 days.Under these conditions,the optimal biomass and total phenol yield were 6.95±0.68 g/L and 3278.44±226.25μg/g DW.3.Enhanced production of phenolic in CCHRCS elicited by UV-B radiation and its regulation mechanism.(1)The optimal induction time of flavonoids in the 42-day-old CCHRCS was 4 h.Under this condition.Under the optimal condition,the resulting flavonoids yield was 414.95±50.68μg/g DW,which was 1.49-fold higher relative to control(278.49±28.36μg/g DW).The optimal induction time of cajaninstilbene acid in the 42-day-old CCHRCS was 10 h.Under the optimal condition,the resulting cajaninstilbene acid yield was 6566.01±702.14μg/g DW,which was 2.31-fold higher relative to control(2842.43±162.57μg/g DW).(2)By determining the contents of three oxidation products and the activities of three typical antioxidant enzymes in CCHRCS before and after UV-B induction,it was confirmed that CCHRCS showed significant oxidative stress defense response to UV-B induction.(3)The phenotype of CCHRCS before and after UV-B induction was observed by scanning electron microscopy(SEM),and it was found that the 42-day-old CCHRCS was severely damaged by UV-B irradiation.In addition,UPLC-Qq Q-MS/MS analysis method was used to detect the content of salicylic acid in CCHRCS before and after UV-B treatment.It was found that the content of cajaninstilbene acid in CCHRCS before and after UV-B treatment was significantly higher than that in control group except 24 h(9.93±2.71 ng/g FW).It was confirmed that UV-B radiation can regulate plant secondary metabolism by triggering the wound/defense signaling pathway.(4)The results of gene transcription and expression analysis of related flavonoids biosynthetic enzymes by q RT-PCR showed that CHS,IOMT,I3’H and IFR are genes closely related to UV-B-induced regulation of flavonoids biosynthesis in CCHRCS.The results of gene transcription and expression analysis of related cajaninstilbene acid biosynthetic enzymes by q RT-PCR showed that PAL2,4CL1,4CL2 and STS1 are genes closely related to UV-B-induced regulation of cajaninstilbene acid biosynthesis in CCHRCS.In conclusion,this study successfully established a UPLC-Qq Q-MS/MS analytical method for accurate tracking and trace analysis of 11 phenolic active components;A C.cajan hairy root system with high yield of phenolic secondary metabolic active components was successfully established.The root high-efficiency culture system provides a theoretical basis for the large-scale production of phenolic compounds by using the CCHRCS;The use of UV-B radiation not only enhances the yield of phenolic active components in CCHRCS,but also initially to clarify the internal mechanism of UV-B regulation of phenolic biosynthesis in CCHRCS,to provide a theoretical basis for high-yield phenolic compounds using metabolic engineering techniques in the future. |
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