| Pear is a fruit tree widely cultivated in the most contries in the world. Apple stem pitting virus (ASPV) is very popular virus infecting pear trees and can result in losses in quality and yield of pear. Cultivation of virus-free seedlings and virus-resistant cultivars are effective measures for the control of virus disease. In recent years, the application of molecular technique using coat protein gene of viruses was proved to be an efficient measure for genetic improvement of plant resistance to viruses. In this paper, ASPV from Pyrus pyrifolia cultivated in China was tested by molecular techniques and isolated by mechnically inoculated onto herbaceous plants. The leaf explants of Nicotiana occidentalis were transformed with Agrobecterium tumefaciens EHA105 containing the recombinant plasmid of cp gene from ASPV. The aim of this study was to establish the a system for obtaining virus-resisitant clones. The results obtained are listed as followings:(1) ASPV was tested from Pyrus pyrifolia in China. A total of 52 samples collected from Fruit tree and mulberry silkworm institute of Hubei agricultural scientific academy were tested for ASPV by TC-RT-PCR. Results showed that the virus specific products with the expected size 316 bp were amplified from 33 samples, which account for 63.5% of tested samples. ASPV from 19 samples collected from Hubei agricultural scientific academy and 10 samples maintained in the green house were inoculated on the by mechanically inoculation, and 9 and 10 samples from these two different sources induced ASPV-specific symptoms on leaves of Nicotiana occidentalis, respectively. All symptom showing herbaceous plants were re-tested for ASPV by TC-RT-PCR. The symptoms induced by the virus on the leaves of Nicotiana occidentali included white necrotic spots, black necrotic spots along leaf vein, chlorosis and vein yellowing. In addition, ASPV from a few samples did not induce obvious symptoms on the leaves of Nicotiana occidentalis.(2) The plant expression vector of cp gene from ASPV was constructed and transformed into Nicotiana occidentalis by Agrobecterium-mediated transformation. The complete cp gene f.rom ASPV isolate 6-1-13sequence was determined Then, the target DNA fragments were ligated into plant expression vector pCAMBIA1301, the constructed plasmids (pCAMBIA1301-CP) were transformed into E.coli DH10B. The plasmids containing inserted cp gene were identified by PCR and digestion -with BamHâ… and Smaâ… , BamHâ… and Sail. The leaf explants from healthy tobacco plants with 5-6 leaves were transformed with Agrobecteriurn tumefaciens EHA105 containing the recombinant plasmid (pCAMBIA1301-CP). By the transformation system of tobacco was modified, the concentration of Hyb and Agrobecterium tumefaciens, and the time of disc treatment with the EHA105 containing the recombinant plasmid were optimized. Genomic DNA was isolated from the differentiate plants of Nicotiana occidentalis transformed with recombinant plamids and preceded by PCR tests based on ASPV cp gene. Products with the expected size were amplified from 88 tobacco plants, among which 68 plants contained the positive stand and 20 plants contained the negative strand. The results indicated that ASPV cp gene was successfully transformed into tobacco plants.
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