| Tetrastigma hemsleyanum Diels & Gilg ex Diels(T.hemsleyanum)belongs to the genus Tetrastigma of Vitaceae,which is a rare medicinal plant in China.Its tuberous roots are used as Chinese medicine and have anti-tumor effect,etc.Because of its slow growth in natural environment and long-period of root formation,it is difficult to meet the demand of medicinal materials market.On the other hand,endophyte is a kind of microbial group,which exists in plants and has various kinds.In the process of long-term evolution,endophyte has beneficial relationship with host plant mutually.Some endophytes can promote the growth,improve the disease resistance,salt resistance and drought resistance of the host.Some endophytes were isolated from the root tuber of T.hemsleyanum in our laboratory previously.The endophyte named as TH15 can promote the growth of T.hemsleyanum,but its molecular mechanism is unclear.In this study,we first confirmed that the endophytic fungus TH15 belongs to genus Fusarium of family Tuberculiaceae based on its molecular characteristic sequence.Futher,the whole genome were sequenced by shotgun method based on Hiseq + pacbio sequencing platform.The active components in fermentation solution(metabolite)of TH15 were classified and identified by HPLC-MS plus NMR analysis.Meanwhile,the content of zearalenone(ZEN)in TH15 was detected by ELISA.Finally,different concentrations of ZEN were added to MS medium to culture plantlets of T.hemsleyanum,cucumber and maize seeds,and observed their effects on the growths of plantlets and seeds.The results are as follows:(1)The genome size of TH15 is 48.74 MB,and GC content is 47.53%.28216 protein coding genes are predicted,and the total length of protein coding gene sequence is 14200482 bp.The predicted repeat sequence is 1187939 bp,and the content of repeat sequence is 2.58%.The predicted number of non coding r RNA is 66,the number of t RNA is 317,and the number of nc RNA is 356.In addition,in the analysis of carbohydrate active enzymes(cazy),the number of GHS was the highest and is 348,followed by 135 CEs,114 AAs,87 GTs and 27 PLs.According to the KOG classification,10.02% of genes are related to carbohydrate metabolism;7.97% of genes are involved in cell transport,biosynthesis and catabolism of secondary metabolites;6.29% of genes are related to amino acid transport and metabolism;3.23% of genes are involved in DNA replication,transcription and energy production,etc;43.98% of genes are divided into regions with unknown.Compared with KEGG database,the genome sequences have 48 metabolic pathways,including protein signaling pathway;genetic information replication and repair;amino acid metabolism;coenzyme and vitamin metabolism;and terpene and polyketone metabolism.(2)Using HPLC-MS and NMR method,the primary and secondary metabolites in the fermentation solution(metabolites)of Th15 were identified qualitatively and quantitatively,which included alkaloids,terpenes,amino acids,indole-3-acetic acid(IAA),indole-3-butyric acid(IBA),gibberellin(GA),abscisic acid(ABA),salicylic acid(SA),zeatin(ZT).Besides,the content of zearalenone in TH15 was 1.70±0.02μ/g?FW by ELISA method.(3)Comparing with the control groups,plantlets of T.hemsleyanum culutred on MS + IAA 0.5 mg/L + 6-BA 0.5 mg/L + 100 m L/L fermentation solution and MS +IAA 0.5 mg/L + 6-BA 0.5 mg/L + ZEN 0.1 mg/L,their seedling height,root length,fresh weight and number of fibrous roots were better after 30 days.In addition,Comparing with the control groups,cucumber and maize seeds cultured on 1/2 MS + 6 g agar + 15 g sucrose + 0.1 mg/L ZEN medium,the seedling height,root length and fresh weight were better after 7 days.The results of this study provide an important basis for further exploring the molecular mechanism of endophytic fungus TH15 promoting the growth of T.hemsleyanum. |
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