| Epichloae endophytes, including asexual(Neotyphodium spp.) and sexual (Epichloe spp.) are symbiotic with gramineae plants. In this study, we use strains obtained from Roegneria origined in China to detect the NRPS genes by the primers designed by Johnson and ourself. Then, we design the primers of perA genes by the conservative section NRPS8, clone the per A gene and detect the distribution of endophytic fungi in strains. At last, we verify the endophyte with host-depengdent features by PCR methods.We collect 200 plants with stroma from 13 collections of 5 cities (Nanjing, Suzhou, Hefei, Lu’an, Hangzhou) in 3 province of Jiangsu, Anhui, Zhejiang. The plant was identified as Roegneria kamoji (Ohwi) Keng and Chen. We isolated 80 strains and choose 20 strains to conduct the morphological characters. The colonies on PDA were white and cottony with brown reverse in the centre and light tan at margin. Their growth rates ranged from 35.5 to 67.5mm after 21d growth. Conidia were ellipse and reniform,3.6±0.6-5.8±1.2 μm in length and 2.2±0.1-3.5±0.8μm in width. Conidiophores were bottle-shaped stems, hyphae, solitary, sepatate usually the base or near base of hyphae,14.3±2.3-22.7±2.4μm in length,1.4±0.4～3.2±1.1μm in width. They were identified as E. yangzii.In this study, we used improved method for genomic DNA extraction from slow-growing fungal endophytes. The improved QS method was achieved by using plate-grown mycelia as the starting material. This new method was in virtue of simple equipment, easy operation, rapid and safe extraction, and low cost. The whole extraction process could be accomplished in 40 minutes. Then DNA was used as template to detect the NRPS genes of the endophytes. The resulted sequences were blasted in NCBI. The quality of DNA samples from this method could be used for subsequent research.We design primers based on the conservative amino acid sequence of Ferrichrome siderophore through CODEHOP online website. Then, we obtain approximately 600bp of the target fragment and similar with the sidC sequence. This shows our strans of E. yangzii has the potential ability of the synthetic Ferrichrome siderophore;We amplified NRPS genes by specific primers of NRPS genes. All NRPS genes spread widely in E. yangzii strains. NRPS2, NRPS5, NRPS6, NRPS7, NRPS8 distribute in almost all E. yangzii strains. There are some differences in the distribution of other NRPS genes in the tested strains. The sidC gene can be detected in some strains. There are also some different in the four strains from the same location. The existence of these NRPS genes indicates that, in our strains of E. yangzii there is a wealth of NRPS gene resources.In this study, we amplified three fragments about 3kb by the three primers that design by the analyses of E. festucae E2368. Then, we connect the fragments and confirm it as perA gene; the size is about 8.5kb. Domain analysis showed the fragment encode a pair of module NRPS. Three pairs of specific primers to investigate the distribution of per A in endophytic fungi, further predict the capacity of peramine synthesis in E. yangzii strains.We first amplified the NRPS8 gene from Reogneria, Calamagrostis, Melica and Poa, and verify that the NRPS8 gene is only existed in the endophyte and the plant with endophyte. We found that the specific gene of NRPS8 could be detected in the metagenomic DNA of endophyte presence or absence. By the metagenomic detection, we can prove that endophytic fungi exist only in its symbiotic host body, which means host-dependent. |
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