Molecular Variability Of Apple Stem Grooving Virus And Apple Cholorotic Leaf Spot Virus

The apple stem grooving virus(ASGV) and apple cholortotic leaf spot virus (ACLSV) are important viruses in apple and pear trees which can cause sereious effect on the growth of fruit trees and the quantity, quality of fruit. The biology, serology and molecular variability of ASGV and ACLSV were studied in this reaserch. The aim of our studies was by analyze to study the relations in biological diversity, serological difference and molecular variability on the molecular level the relations in molecular variability, biological and serological characters among different isolates.The results are described as following:1. Twenty-three isolates of apple stem grooving virus were obtained from pear and apple by mechanically inoculating on Chenopodium quinoa and ELISA confirmation. Specific fragments were amplified from all these samples by tube capture reverse transcription-polymerase chain reaction (TC-RT-PCR). These PCR products showed three type bands with different migration rate (500bp, 530bp and 600bp) on 5%PAGE. The PCR products of three isolates P-L4, P-6-1-17 and P-3-2-67 from pear with different migration rates were cloned and sequenced. A BLAST search result revealed that these three isolates had 92.2%, 90.4% and 88.4% nucleotide sequence similarity with the 3' ends of P-209, an isolate from apple. And the nucleotide sequences among these three isolates also showed some difference. The similarities of P-L4 and P-3-2-67, P-L4 and P-6-1-17, P-6-1-17 and P-3-2-67 were 90.4%, 95.5% and 88.6%, respectively.2. Four isolates of Apple stem grooving virus, P-6-1-17, P-4-1-69, P-L2 and P-D-21 from pear, which displayed different reactions in biological and serological tests, were studied for its molecular characteristics. RT-PCR products of 3 ' end of four isolates were cloned and sequenced. The sizes of cloned fragments are 1089nt from P-6-1-17 and P-L2, and 1069nt from P-4-1-69 and P-D-21, respectively, which include 714nt CP coding region. The similarities of nucleotide and deduced amino acid sequences of CP genes among these isolates are 89.8% to 96.4% and 94.9% to 97.9%, respectively. The sequence data obtained in the study were compared with previously reported sequences of ASGV isolates and sequence variant species from different sources. Results suggested that isolates P-6-1-17, P-4-1-69, P-L2 and P-D-21 should be divided into two different subgroups. P-L2 showed different reactions in biological test and its CP gene also had a relatively high variability.3. Thirteen isolates of apple cholortotic leaf spot virus were obtained from pear, apple and peach by mechanically inoculating on Chenopodium quinoa and ELISA.PAS-ELISA and DAS-ELISA were used to detect these 13 isolates, in the former method one peach isolates (HBP) was positive reaction and in the latter method three peach isolates were positive reactions (HBP PE-T-5 和 PE-5) . Otherwise, when RT-PCR and TC-RT-PCR were used to detect these 13 isolates, specific products of 358bp fragment were obtained in all these isolates.4. Two isolates from peach and apple, HBP and ACLSV-C displayed systemic chlorotic symptoms on Chenopodium quinoa and Ch.amaranticolor had relations in serological tests. RT-PCR products of two isolates were cloned and sequenced. The sizes of cloned fragments are 1768nt from HBP(GenBank accession numer: AY728180) and 1751nt from ACLSV-C, respectively, which include 714nt CP coding region. The similarities of nucleotide and deduced amino acid sequences of CP genes among these isolates are 87.8% and 95.9% respectively. The sequence data obtained in the study were compared with previously reported sequences of ACLSV isolates from different sources. The similarities between HBP and SX/2 were high but the latter did not react (or reacted weakly) with some moloclonal and polyclonal antibodies prepared against ACLSV isolates from apple and cherry.5. A fragment 485nt corresponding to 83.3% of the 3'end coat protein gene of P-4-1-69 isolates of apple cholortotic leaf spot virus from pear was obtained. The similarities of nucleotide and deduced amino acid sequences of 3'end CP genes among P-4-1-69, HBP and ACLSV-C isolates are 86.2% to 99.8% and 96.2% to 100.0%, respectively. The sequence data obtained in the study were compared with previously reported sequences of ACLSV isolates from different sources. Results suggested that isolates P-4-l-69 HBP ACLSV-C Kuerle and MP-CI should be divided into two different subgroups.6. The result of SDS-PAGE of prokaryotic expression of the coat protein of HBP demonstranted that this gene was expressed in E.coli, the molecular weight of the fusion protein was 47.6kDa. Western-blot examination identified the gene was expressed rightly, and the fusion protein was immunogenic.