Due to its accuracy, sensitivity and high throughput, real time quantitative PCR (RT-qPCR) has been widely used in analysing gene expression. The quality of data from such analyses is affected by the quality of reference genes used. Expression stabilities for nine candidate reference genes widely used in soybean were evaluated under different stresses in this study. Our results showed that EF1A and ACT11 were the best under salinity stress, TUB4, TUA5 and EF1A were the best under drought stress, ACT11 and UKN2 were the best under dark treatment, and EF1B and UKN2 were the best under virus infection. EF1B and UKN2 were the top two genes which can be reliably used in all of the stress conditions assessed.

Leaf rust caused by Puccinia triticina is one of the main diseases affecting wheat (Triticum aestivum) production worldwide. Calmodulin (CaM) was found involved in the early stage of signal transduction pathway in response to P. triticina in wheat. To study the function and molecular mechanism of calmodulin (CaM) in signal transduction of wheat against P. triticina, we cloned a putative calmodulin-binding transcription activator (TaCAMTA4), and characterized its molecular structure and functions by using the CaM-encoding gene (TaCaM4-1) as a bait to screen the cDNA library from P. triticina infected wheat leaves. The open reading frame of TaCAMTA4 was 2505 bp encoding a protein of 834 aa, which contained all the four conserved domains of family (CG-1 domain, TIG domain, ANK repeats and CaM-binding domain). TaCaM4-1 bound to TaCAMTA4 by the C-terminal CaM-binding domain in Ca²⁺-dependent manner in the electrophoretic mobility shift assay (EMSA). Bimolecular fluorescence complementation (BiFC) analysis indicated that the interaction of TaCAMTA4 and TaCaM4-1 took place in the cytoplasm and nucleus of epidermal leaf cells in N. benthamiana. The expression level of TaCAMTA4 genes was down-regulated in incompatible combination after P. triticina infection. Furthermore, virus-induced gene silencing (VIGS)-based knockdown of TaCAMTA4 and disease assays verified that silencing of TaCAMTA4 resulted in enhanced resistance to P. triticina race 165. These results suggested that TaCAMTA4 function as negative regulator of defense response against P. triticina.

The function of callose and its deposition characteristics at phloem in the resistance to the long-distance transportation of Soybean mosaic virus (SMV) through phloem was studied. Two different methods of SMV inoculation were used in the study, one was direct friction of the virus on seedling leaves and the other was based on grafting scion and rootstock to create different resistance and sensitivity combinations. Veins, petioles of inoculated leaves and rootstock stems were stained with callose specific dye. Results from fluorescence microscope observation, pharmacological test, and PCR detection of SMV coat protein gene (SMV-CP) showed the role of callose in long-distance transportation of SMV through phloem during infection of soybean seedlings. When the inhibitor of callose synthesis 2-deoxy-D-glucose (2-DDG) was used, the accumulation of callose fluorescence could hardly be detected in the resistant rootstocks. These results indicate that callose deposition in phloem restricts the long-distance transport of SMV, and that the accumulation of callose in phloem is a main contributing factor for resistance to this virus in soybean.