| Objective:Aquilaria sinensis (Lour.) Gilg, also known as, Nverxiang and Tuchenxiang. It belongs to the Thymelaeaceae Aquilaria. It is the only species in China production of eaglewood, was listed as the two national endangered plant Chinese eaglewood is produced resinous wood Aquilaria sinensis from damage or physical biological chemical stimulus, is the traditional rare medicinal herbs and natural flavor, and has very high medicinal value and commercial value, but its natural accumulation process takes a long time and the current sink for decades, incense forming process and its mechanism has not been clarified yet establishment and aloes, seriously restricted the stable and efficient Aquilaria sinensis artificial Edgeworthia technology production, supply, prices rising situation aloes, appear on the market. Many studies confirmed the formation of aloes and endophyte effects related to, but are based on the traditional methods of laboratory culture. This study used culture independent molecular biological methods:PCR- denaturing gradient gel electrophoresis that the PCR-DGGE technology, and the terminal restriction fragment length diversity is T-RFLP (Terminal-restriction fragment length polymorphism, T-RFLP) comparison of the technology of Aquilaria sinensis not knot colony structure of white wood and sweet incense wood endophytes, rhizobacteria Aquilaria sinensis sweet to investigate the effect of internal, expect to find more and better able to promote sweet microorganisms, provide scientific basis for the establishment of artificial sweet method of high quality.Method:In this experiment, Aquilaria sinensis not knot fragrant white wood andsweet parts as materials, the endophytic bacteria by using PCR-DGGE, T-RFLP technology, community structure and diversity of endophytic fungi were studied to investigate the incense, before and after the change of Aquilaria sinensis, endophytic fungi. Specific methods are as follows:1. sample collectionIn order to avoid exogenous microbial contamination, with growth cone sterilized remove sites of sampling based surface, then quickly into a sterilized bag for collecting, transport back to the laboratory -20 ℃ preservation reserve.2. The sample processingEach samples in ultra clean sterile environment dry surface after disinfection. In order to avoid the surface microbial DNA pollution, ensure the extraction of nucleic acid from inside the plant, scraping with a blade to the sample surface layer microstructure, shaving Edgeworthia group samples will decay wood and may be white wood joint. Will handle a good sample of aseptic environment liquid nitrogen at the end of the research, in a sterile centrifuge tube standby.3. Extraction and purification of total DNA of each sample DNA extraction by plant genomic DNA extraction kit, extraction methods according to kit instructions in operation.4. Total DNA concentration and purity detection Using 1% agarose gel electrophoresis to detect the DNA fragment size, and Using protein- nucleic acid instrument measuring the concentration of DNA.5. DGGE analysis of endophytes Taking the sample total DNA as template, the universal primer respectively with GC clip of fungi and bacteria were amplified, DGGE separation of amplified products after 1% agarose gel detection.6. Endophytic bacteria T-FRLP Taking the sample total DNA as a template, with FAM fluorescent groups of bacteria and fungi universal primers were amplified respectively by.1% agarose gel electrophoresis of PCR amplified products were purified, gel recovery kit. The purified PCR products were digested by restriction endonuclease. The digested products commissioned a Shanghai Genecore Biotechnologies Company for gene scanning.Results:Whether it is sweet or not sweet group, DGGE fungi, bacteria isolated from the strip of rich diversity, and on the whole, sweet group of endophytic bacteria diversity was higher than that of sweet group. Obtain multiple bacterial, fungal flora of T-RF fragment of T-RFLP scan. Previous reports in the literature is more endophytic fungi on the impact of node fragrant the related report rare endophytic bacteria, this research will Aquilaria sinensis of endophytic bacteria also as the object of study, the results showed bacterial.colony structure also has the huge change in students before and after the sweet.Conclusion:agarwood formation and normal white wood there are a large number of endophytic bacteria, compared to the node incense before, number and structure of endophytic bacteria arechanged, the concrete manifestation is more diversity, infer, endophytic fungi and bacteriaare involved in Aquilaria sinensis sweet... |
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