| Plant diseases is one of the main restrict factors for agricultural production. TuMV is an important phytopathogen, widespread all over the world. According to the virus disease investigation in 28 countries and districts, TuMV has become the second important pathogen that damages the vegetables, just after CMV. TuMV is the primary pathogen of Cruciferae virus disease in China, which is widespread and destructive, leading to the fatal economical loss in Cruciferae production. This virus is well studied in China, due to its importance.The mechanism of phytoresistance is quite complicated;the knowledge of interaction between plants and pathogen is poor;and the general breeding approach has its own limitations;as a result, phytoresistant breeding remains a big problem in agricultural production. In the last decade, several phytoresistance-relative genes have been cloned, consequently, the study on molecular mechanism of interaction between plants and pathogens has made great progress, which is beneficial and promising for the application of resistance-relative genes in production.This research took advantage of collected mustard species and molecular tools. The resistance-relative genes were cloned, examined by Northern blot;the state of disease was investigated and tested using EALAS after inoculation of virus. It was aimed to obtain a molecular method to rapidly identify the resistance of mustard to TuMV and lay a basis for further study.We used the 18 mustard species collected from Zhejiang, Shanghai and Sichuan as material and obtained one special band of 641bp length, via PCR amplification, in species that were highly resistant in field. According to the pertinence between this special band and the resistance of each mustard species to TuMV, this fragment was supposed to be relative to the resistance of mustard to TuMV. Primers were designed according to the sequencing result of this special fragment and the full gene of 1039bp length was cloned with RACE. Blast on NCBI gave the result that no gene sequence has a similarity more than 50% with this target gene. This gene might be an unknown gene.After extraction, RT-PCR examination and Northern Blot of RNA from all the tested material, we discovered, this gene was highly expressed in species no.3, 5, 6, 7, 10, 11 and 13, while lowly expressed or not expressed in other species. It was consistent with the performance in field, friction inoculation in glasshouse and examination of ELISA. In the highly resistant species, this gene was highly expressed, the plant was highly resistant, resistant or disease-tolerant, while in the lowly resistant or susceptible species, it was lowly expressed or not expressed, the plant was disease-tolerant or sick.Meanwhile, the tested material was planted in grasshouse without aphids. At the four-leaf stage, each mustard species was inoculated with TuMV, with ten repeats each, to identify the resistance to. TuMV. All the material were investigated by the incidence of disease and disease index after ten days. The investigation showed, there were 7 susceptible species (no.2, 8, 9, 12, 15, 16 and 17) , 7 disease-tolerant species(no.l, 4, 6, 11, 13, 14 and 18), 3 resistant species(no.3, 7, and 10) and 1 highly-resistant species(no. 5) in the 18 mustard species, without any immune species. It was consistent with the expression level in the species. The concentration of TuMV in the material was measured by ELASA after ten days' inoculation and the OD^swas obtained for each species. The result indicated, the concentration of TuMV is obviously lower in the species with high target gene expression level, and vice versa. It was consistence with the result of disease state investigation and our speculation.This gene was defined as relative to the resistance of mustard to TuMV. The resistance of mustard to TuMV can be measured by molecular detection of this gene, which is an easy and shortcut way and has wide application value. |
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