| Taxus cuspidata mainly grows in Laoyeling,zhangguangcailing and Changbai Mountains of Jilin Province,China.taxoids are the main secondary metabolites of Taxus,among which paclitaxel is the most representative of taxoids,which is widely recognized as a broad-spectrum anticancer drug in the world.The growth of T.cuspidata is very slow and the content of taxoids is low,which can not meet the demand of domestic and foreign markets.Therefore,the application of biotechnology to obtain taxoids has become one of the main technical means to solve the problem of taxoids shortage.It has the advantages of short culture cycle,artificial control of culture conditions,not affected by seasons and regions.At present,it is a feasible key technology to improve taxoids production from Taxus tissue culture by induction technology,and using LED light has become a promising technology to improve the production of secondary metabolites in plant tissue culture system.In this study,the callus culture system of T.cuspidata was optimized and established.Five kinds of LED lights with different light quality(red light,blue light,green light,red blue green mixed light and white light)were used to irradiate the callus.The dark culture was used as a control,and seven taxoids(paclitaxel,bacatine ?,10 deacetylbacatine ?,10-deacetyltaxol,cephalomannine,7-xylosyl-10-deacetyltaxol and 7-epitaxol)were analyzed under different light quality The accumulation and change were studied,and the expression level of six genes(TXS,T7?OH,DBAT,ACL,BAPT and DBTNBT)closely related to paclitaxel biosynthesis in callus was analyzed by qRT-PCR,which provided a basis for the establishment of callus culture system of T.cuspidata scientific basis.The main results are as follows:1?The callus culture system of T.cuspidata was optimized and establishedTake the young stem of T.cuspidata as explant,wash it under tap water for 4 hours,transfer it to aseptic operation platform,sterilize it with 75%alcohol for 30 seconds,then sterilize it with 0.1%HgC12 solution for 15 minutes,dry the surface water of stem section with aseptic filter paper,cut off the leaf with scissors,cut the stem section to 0.5 cm length,and inoculate it at MS+2 mg/L NAA+0.1 mg/L callus was induced on KT medium.After 30 days of induction,the callus was stripped to MS+2 mg/NAA+0.1%activated carbon medium for callus proliferation.2?Effect of different light quality on the accumulation of taxoidsDifferent light quality(red,green,blue,mixed and white)led lights were used to irradiate the callus of T.cuspidata.Dark culture was used as control,and 7 taxoids were analyzed by UPLC-MS/MS.the results showed that different light quality had different effects on the accumulation of taxoids.However,the accumulation of taxoids in the callus of T.cuspidata could be enhanced by 5 light qualities,and the best light quality was determined,which could increase the total taxoids content by 2.17 times and the paclitaxel content by 2.84 times.In addition,after 30 days of red light irradiation,it was found that the content of taxoids in the callus of T.cuspidata increased gradually and then decreased with the increase of time.At 25 days,the content of all target compounds was the highest,the total content of taxoids was 61837.78 ± 4893.59 ng/g DW,and the content of paclitaxel was 21385.07± 2484.95 ng/g DW.3?Effects of different light quality on the expression of genes related to the biosynthesis of taxoidsqRT PCR was used to study the expression of six genes related to taxoids biosynthesis in T.cuspidata callus under different light quality.In control with dark culture,it was found that almost all of the light could promote the expression of six genes,which was consistent with the effect of different light quality on the accumulation of taxoids compounds,indicating that different light quality stimulated the transcription expression of these taxoid biosynthesis genes,thus promoting the synthesis and accumulation of active components of taxoids secondary metabolism.In particular,under red light,except T7?OH,the expression of other genes reached the highest level,especially ACL gene showed the highest transcription abundance under red light,up to 12.51,which may be the internal reason for the accumulation of taxoids under red light.After 30 days of red light irradiation,most of the genes showed a trend of gradually increasing and then decreasing,which was the same as that of taxoids.It is suggested that the simultaneous up regulation of these synthetase genes results in the induction and enhancement of taxoids biosynthesis and accumulation in T.cuspidata callus by red light.Among them,TXS,DBAT and ACL genes had the highest expression level on the 21st day,T7?OH,BAPT and DBTNBT genes all reached the highest level on the 13th day,and the highest expression level of these genes was earlier than the time point(25 days)required for the highest accumulation of taxoids,which was in line with the metabonomics rule that gene expression was earlier than product synthesis.At the same time,this study preliminarily confirmed that TXS,T7?OH,DBAT and ACL may be the potential key genes of taxoids biosynthesis.In addition,ACL gene transcription abundance(up to 36.08)in 21 days was significantly higher than other genes,which indicated that ACL gene was more sensitive to taxoids synthesis induced by red LED.This study confirmed that the taxoids accumulation in the callus of T.cuspidata could be effectively enhanced by red light irradiation.This method is simple,pollution-free,low cost,and has potential application value.In addition,the intrinsic mechanism of light quality regulation on taxoids biosynthesis was preliminarily clarified in this study.It was found that ACL gene was more sensitive to red light response,which was helpful to the synthesis and accumulation of taxoids compounds,and provided a theoretical basis for the future use of metabolic engineering technology to produce high-yield taxoids. |
PDF Full Text Request