| Objective: 1.Screening and optimization of optimal media formulations for inducing B.striata callus.2.Observing the process of symbiotic culture of B.striata callus and endophytic fungi,and analyze the effect of endophytic fungi on the synthesis of secondary metabolites.3.Detecting the SNP in callus during different stages of suspension culture.Methods: 1.To optimize B.striata cinduction medium by orthogonal experiment method: the mediums were designed by using two-factor and three-level orthogonal experiments,the obtained sterile seeds were transferred into these nine mediums,and the B.striata callus induction rate was calculated after 50 days of culture.To optimize callus proliferation medium,Box-Benhnken response surface method was applied as follow: according to the BBD test design principle,a response surface test was designed.the BBD test factors were determined to be 6-BA,2,4-D,and NAA at different concentrations,and the response value was the callus proliferation rate.Loose and bright yellow callus was selected from the induction medium,which was inoculated in 13 kinds of proliferation medium.And callus proliferation rate was calculated after 20 days.2.Composition of secondary metabolites were detected after co-cultured of endophytic fungi and callus.A total of 8 strains of endophytic fungi with different growth status and colors in the PDA medium were selected and inoculated to the B.striata callus with the optimal medium of callus proliferation.During the culture and growth process,the growth state of the endophytic fungi and the browning degree of the B.striata callus and the contact degree between the B.striata callus and the endophytic fungus in the co-culture state was observed.The callus was removed from the co-cultivation medium on the 3rd,6th,and 9th days,and the volume was adjusted to 5 mL after extraction with 70% methanol,then the change of metabolites during the 9 days of co-culture was measured by HPLC.3.The aforementioned induced the B.striata callus was transferred to a liquid medium for suspension culture,from the beginning of the inoculation to 45 days,callus cells were collected every three days to extract their total RNA.Take an equal amount of each RNA sample to mix as a pool for full-length transcript sequencing of the third generations,which was produced a reference for following analyzing.Each RNA sample was wend on to conduct second-generation RNA-Seq sequencing,and the sequencing results were blasted to the reference to detect SNPs.Results: 1.Using seeds as explants,the orthogonal culture method was used to obtain the optimized culture medium for B.striata callus induction culture: MS+1.0 mg/L 6-BA+2.0 mg/L 2,4-D+6.0 g/L agar +30 g/L sucrose.Based on the orthogonal test-induced callus,according to the Box-Benhnken(BBD)test design principle,a three-factor with three-level method was used to conduct a combination experiment of different concentrations of three hormones.The results showed that the optimal medium for proliferation induction obtained by regression model analysis was MS+0.801 mg/L 6-BA +1.192 mg/L 2,4-D+0.724 mg/L NAA+ 6.0g /L agar +30 g/L sucrose.2.The callus with good growth state in the third subgeneration were selected for co-culture with endophytic fungi,and the contents of secondary metabolites were determined on d3 dai,6 dai,and 9 dai,respectively.Through the determination of secondary metabolites,it was found that the endophytic fungi with accesion number of 1-56 had the greatest influence on the content of p-hydroxybenzyl alcohol,and on the 6 dai,the content of p-hydroxybenzyl alcohol was 2.5 times that of the control group.Endophytic fungi numbered 1-56 and 3-4 had a significant effect on the content of dactylorhin A.Endophytic numbered 1-42 fungi on content of militarinewas slightly higher than that of the control group on 9 dai.Endophytic fungi numbered 3-4 had the most significant effect on the content of coelonin.3.Samples of 10 timepoints in suspension culture were selected for RNA-Seq sequencing in the third and second generation,and 241,570 SNP sites were detected,which were mainly divided into two forms: transition and transversion.The number of transition and transversion types were 78,184 and 41,062,respectively.The largest number was C/T type,which reached 44248.From the quantitative analysis,on the third day,the lowest number of Homo sites were 18411,and the highest number was 41430 on the 33 rd day,the average number was 29920.Respectively,the lowest and highest number of Heter sites were 5,340 at day 3 and 14,711 at day 9,respectively.And the average was 10007.Conclusion: In this study,the induction medium and subculture medium of B.striata callus are successfully optimized by orthogonal test and response surface test,respectively.In addition,8 strains of endophytic fungi for co-cultivation with B.striata callus are selected.Through the determination of the content of secondary metabolites,the endophytic fungi which could stimulate the significant changes of the secondary metabolites.SNP data are compared and analyzed. |
PDF Full Text Request