Research On Fast Detection Of Apple Stem Pitting Virus In Pyrus Pyrifolia And Clone And The Prokaryotic Expression Of Its Coat Protein Gene

Pear is the traditional fruit in China and its planting area and total yield rank the first in the world. But the poor average yield and quality of pears seriously affected the Chinese pears export business. Virus disease is one of the important factor which affects the yield and the quality and lead to the serious reduction of output in pears. Apple stem pitting virus (ASPV) is one of three most important latent viruses in Pyrus pyrifolia and is the representative specie of Foveavirus. It specificly infects apple and pear, causing peak dieing, inner tegument necrotic, graft declining, leaf epinasty in the sensitive rootstocks which reduce the yield and the quality of pear, influencing seriously the development of pear industry.In present, the cultivation of virus-free varieties is the effective way to reduce the losses caused by ASPV. The virus detection is the critical step in the cultivation and propagation of virus-free varieties. However, isolation and purification of ASPV is very difficult because ASPV is little, and hydroxybenzenes is abundant in apple and pear tree tissues. The antibody for detecting ASPV has not been obtained in China so far.In order to set up rapid detection techniques of ASPV, three methods including biology, serology and molecular biology were used in our studies. The coat protein gene of ASPV in the sample 6-1-1.3 was cloned, and its complete nucleotide sequence were obtained. Based on this, its prokaryot expression was carried out in E.coli and the product was identified by western-blot. The results are described as following:Biological index: in the green house, with indexing of herbaceous indicators, ten samples which are positive by the genenral PAS-ELISA detection caused the irregular spots on the leaves of Nicotiana occidentalis '37B' In addition, 6-1-13, 6-1-103 and 4-2-51, three samples caused vein yellow. With indexing of woody indicators, ten samples caused leaves rolling, stunt, spitting in Pyronia veitchii; chlorotic spots, vein yellow in A20. Results showed the index of indicators of ASPV is accurate.Serological detection: The improved PAS-ELISA, PAS-ELISA and PTA-ELISA methods were applied to detect ten samples. The results showed that adding the second antibody and protein A enzyme conjugates simultaneously can efficiently reduce the nonspecific reaction and then enhance the sample absorbance ratio of positive and negative. The same results were obtained with PAS-ELISA and PTA-ELISA methods. The absorbance ratio of positive and negative samples turn higher as the sample diluted, especially when the sample was diluted 5 times. Overall, the method is safe, rapid andaccurate and is suitable for high throughput detection.Molecular detection: Based on the reported genome sequence of ASPV, we designed the primers to detect ASPV in six samples, 6-1-13, 4-2-51, 4-1-51, 6-1-35, 6-1-73 and 6-1-103 by RT-PCR using dsRNA as template. The expected fragment about 316bp was amplified in all six samples. The corresponding fragment was also amplified by TC-RT-PCR in three samples, 6-1-13, 4-2-51 and 6-1-103, but not in other two samples, 6-1-73 and 6-1-35. Because dsRNA is very stable, RT-PCR using dsRNA as template can detect viruses effecientlv. TC-RT-PCR need not extract nucleic acid, and captures viruses by non-special adsorption of the tube, so it's simple in manipulation and safe to the operator and environment. Both methods are special, sensitive, rapid and accurate and suitable to detect samples on the large scale.Cloning and sequence determining of coat protein gene: Based on the reported genome sequence of ASPV, we designed the primers to amplify the coat protein gene of ASPV in 6-1-13 by RT-PCR using dsRNA as template. The expected fragment about 1328bp was obtained. The amplified product was purified and cloned to pMD18-T and then sequenced. The comparative analysis of ASPV coat protein gene sequences between 6-1-13 and other published isolates showed that ASPV coat protein gene from 6-1-13 shared a homology ranging from 82%-88% with other ten isolates logged on GenBank. 6-1-13 and ST132 has the higest identity of 88% as 6-1-13 and GNKV(?)/34 has the lowest of 82%. On the amino acid sequence level, there also exist a homology of 93%-98% between 6-1-13 and the other 10 isolates. The identity of amino acid between GNKV(?)/34 and it was 93%, the rest was beyond 96%. Phylogenetic tree of nucleotide sequences of 11 isolates of ASPV indicated that ASPV in 6-1-13 had same origin with the isolate GNKIII/45.Prokaryotic expression and western-blot of the coat protein gene of ASPV: pET28c-CP was obtained with link of the targeted gene from T cloning and pET28c. This gene was expressed in E.coli, the molecular weight of the coat protein was 44 kDa. Western-blot examination identified the gene was expressed rightly, and the coat protein was immunogenic.The above results for the first time set up the rapid detection techniques of ASPV in Pyrus pyrifolia, cloned and prokaryoticlly expressed the coat protein gene of ASPV in China. The right expression of the coat protein may not only provid a effctive way to prepare antibody for detecting ASPV, but also highlight the further studies on the function research of this gene, and benefit the coat protein mediated antivirus genetic engineer.