Molecular Detection And Partial Nucleotide Sequence Clone And Genomic Analysis Of Apple Stem Grooving Virus Of Korla Aromatic Pear In Xinjiang

ASGV is one of the most infecting viruses which severely jeopardized fruits. Recently, Kuerle's pear was infected by many viruses including ASGV, which caused a slow decline in the growth and output of apple, as well as the character of fruit. Fruit will become macrocephalic and inedible, losing its commercial value. The purpose of this study was to establish the rapid, efficient and specific reagent system to detect ASGV. At the same time, partial sequence of ASGV from Kuerle's pear has been surveyed and analyzed in order to realize more structure and function of its genes, which will mainly establish theoretical foundation for virus-free breeding and virus-free seedling of Kuerle's pear.Specific fragment has been obtained based on the high quality of total RNA and aligned with the strains of ASGV published on Genbank on the level of nucleotide or amino acid. The result showed lower relation between L , Li-23 and D14455, D16368, but they had high relation with Kuerle's pear,P-209 and AF233298. Nucleic acid spot hybridization (NASH), showing the detection limit 5^-7, and One-step multiplex RT-PCR assay for rapid detection of ASGV have been established.The study included five major conclusions as followed:1. Total RNA was extracted from different tissues of Kuerle's pear using methods improved based on the general ones using in our laboratory. And the methods suitable for different tissues of Kuerle's pear were screened out.2. A specific fragment of about 524bps was obtained by RT-PCR (reverse transcription- polymerase chain reaction) using the templates of total RNA and specific primer. The specific fragment was sequenced by Shanghai Sangon Biological Engineering Technology Corporation. Then it was aligned by BLAST with the sequence NC001749 published representing a homology of 92.75%. Percentage of identity of nucleotide and amino acid sequence between plants of ASGV was obtained.3. The biotin-11-dUTP and DIG-11-dUTP labeled cDNA probe prepared by cloned plasmid and the detection system of ASGV by nucleic acid spot hybridization (NASH) has been established in this experiment. The results showed the NASH detection limit labeled by biotin-11-dUTP was 5^-2 and 5^-3 labeled by DIG-11-dUTP.4. The optimized one-step multiplex PT-PCR detection system of ASGV was established and then the detection of samples of ASGV proved the system was stable and specific. The detection limit was 5^-7, 5⁴ times than that of NASH.