Investigation On DNA Microarray Technology For The Detection And RFLP Analysis Of Flavescence Dorée Phytoplasma

16SrDNA sequence of major representative species of phytoplasma 15 groups were analysed, and primers and probes of DNA microarray were designed in this study, expected that a fast, sensitive and high-throughput approach for the detection of Flavescence dorée (FD) phytoplasma was provided. The fragment (about 290bp) was amplified from phytoplasma with DNA microarray primers, which was consistent with expectant size, the results showed that the reaction system of fluorescent marker PCR was normal; the specific experiments of probes showed that: Phy probe appeared positive signals when detecting only phytoplasma, FD probe appeared positive signals when detecting only 16SrV group phytoplasma, PaWB probe appeared positive signals when detecting only Paulownia witches phytoplasma, the results show that the designed probes were differential; the sensitivity experiments showed that: the minimum target concentrations that could be detected were 103 copies/μL, better than that of the PCR method, and many different phytoplasmas could be distinguished synchronously from hybridization signals by DNA microarray; the stability experiments showed that: the reproducibility and stability is good, DNA microarray within batch and batch to batch can detect significant hybridization signal in the same hybridization conditions, but these signals exhibited respective differences, so only qualitative determination can do; the hybridization system by optimized showed that: the best hybridization temperature was 45℃, hybridization time was 1 hour and formamide was 2.5%, SDS was 0.2%, SSC concentration was 6×SSC. To sum up, a system for the detection of FD phytoplasma were established successfully.The 16SrV group phytoplasmas were amplified with universal primers R16mF2/ R16mR1 and construct recombinant plasmids, these plasmids were identified by restriction enzyme and sequencing. Aliment reports showed that the sequence accord with relevant phytoplasmas sequence in GenBank published. 16SrDNA of 16SrV group phytoplasmas were digested with Xmn I, Xsp I, Taq I and Rsa I 4 restriction enzymes, and electrophoresis results showed that: in addition to, Flavescence dorée-C (FD-C) and Rubus stunt (RUS) phytoplasmas can not distinguish mutually from the specific fragments, the others can do; Taq I was specific for 16SrV-D subgroup phytoplasma and Rsa I was specific for 16SrV-B subgroup phytoplasma.Grape yellow phytoplasma disease were surveyed in Xinjiang, the plants with suspected symptoms were detected by PCR and DNA microarray, the results showed that: grape yellows phytoplasma disease have not been found in Xinjiang region; but Jujube witches'broom (JWB) phytoplasma has been found in Xinjiang Aksu, which and FD phytoplasma belonged to the same group (16SrV). Grape yellows phytoplasma disease have not been yet found, but there were objective conditions for the disease exist in Xinjiang. Therefore, it is necessary to strengthen detection and prevention for grape yellows phytoplasma disease.