Reference Gene Screening And Callose Metabolism-related Genes Expression Analysis By RT-qPCR During Soybean Resistance To Soybean Mosaic Virus Infection

Due to its accuracy, sensitivity and high throughput, real time quantitative PCR(RT-qPCR) has been widely used in gene expression analysis. The quality of data fromsuch analysis is affected by the quality of reference genes used. Expression stabilities fornine candidate reference genes widely used in soybean were evaluated under different fourconditions of stresses in this study. Our results showed that EF1A and ACT11were the bestunder salinity stress; TUB4、TUA5and EF1A were the best under drought stress, ACT11andUKN2were the best under dark (aging) treatment, and EF1B and UKN2were the bestunder virus infection. EF1B and UKN2were the top two genes which can be reliably usedin all of the stress conditions assessed.It has been proven that callose deposition at plasmodesmata is a critical factor inrestricting the cell-to-cell movement of Soybean mosaic virus. On this basis, Jidou7andsoybean virus strains Sc-8and N3were composed of the compatible combination and theincompatible combinations respectively. At different time points after inoculation theexpression of callose metabolism-related genes were analysed, using drawn stableexpression of EF1B and of UKN2as the reference genes and using the mock as control.The results showed that the expression of B1and B2were induced both in the incompatiblecombination and the compatible combination. Callose synthase C7expressed significantlydifferentially in the compatible and the incompatible combination. In the compatiblecombination, no change was observed, but in the incompatible combinations, theexpression began to increase at8h after inoculation and it was54.5times to control at144h after inoculation. The expression tendency of C11and C12were similar in the two kindsof combinations. They decreased slightly in early infection stage (0~12h after inoculation),at96h after inoculation the expression began to increase in the incompatible combination.While in the compatible combination, at96h and144h after inoculation amount recoveredand remained at control levels. The expressions of C1and C2were up-regulated both inthe compatible and the incompatible combinations. During the inoculation process in thetwo kinds of combinations, the expression of C3, C5and C9were remained and a downward trend was showed in C8and C10.It has been demonstrated that callose began to accumulate at plasmodesmata(PD) inthe early inoculation stage in the incompatible combination (2h after inoculation), and itwas not observed in the compatible combination at PD. Subcellular localization ofenzymes suggested that, hydrolase was localized in the vacuole in the incompatiblecombination, and synthase was localized in PD, but in the compatible combination twoenzymes were localized at PD. So the induce of the two hydrolase was unconcerned withcallose accumulation at PD in the incompatible combination. C7was closely related withthe callose deposition at PD, C11, C12may be involved in the late inoculation stage. C1，C2，C3，C5，C9，C8and C10were unconcerned with callose accumulation at PD in theincompatible combination.