Cloning And Preliminary Functional Analysis Of GmCASc Gene In Soybean (Glycine Max(L.) Merrill)

Cytoplasmic-nuclear male sterility (CMS) plays an important role in the utilization of crop heterosis. It is of important significance on the theory and practice to study the genetic base and mechanism of CMS. Based on the differential proteomic studies of soybean cytoplasmic-nuclear male-sterile lines and their maintainer lines, the cloning and preliminary functional analysis of the cysteine protease gene were carried on in this paper.The main results were as follows:1. According to the mass spectrum data of the cysteine protease, a gene highly homology with the sequence of the cysteine protease was found through searching the database of soybean genome. Then the cysteine protease gene was cloned in soybean and named GmCASc.The GmCASc had an ORF of 1101 bp and encoded 366 amino acids. The molecular weight of the deduced GmCASc protein was 40.8 KD and the theoretical pi was 7.49. Homologous analysis of the amino acid sequences showed that GmCASc shared 54%, 52%,56%,44% homology with those of the caspases from Populus trichocarpa, Vitis vinifera, Ricinus communis, Oryza sativa respectively. Bioinformatics analysis indicated that GmCASc had a caspase domain and belonged to CASc superfamily. GmCASc was a non-transmembrane hydropathic protein.2. The expression of GmCASc in different soybean organs and under different stress conditions was analyzed by real time quantitative PCR. GmCASc was found to be expressed in root, stem, leaf and flower of soybean cytoplasmic-nuclear male-sterile lines NJCMS1A, NJCMS2A and their corresponding maintainer lines NJCMS1B, NJCMS2B. The relative expression quantity of GmCASc showed the highest level in the stem. The relative expression quantity of GmCASc in the flower of NJCMS1A was lower than that of NJCMS1B, while the relative expression quantity of GmCASc in the flower of NJCMS2A was higher than that of NJCMS2B. The expression quantity of GmCASc in the leaves acted differently under biotic and abiotic stress conditions. The GmCASc expression was down-regulated under low temperature stress, but accumulated under salt stress, SMV, and mechanical damage treatment. 3. The yeast two-hybrid system was used to study the interations among cysteine protease, V-type H+-ATP enzyme subunit, AMP deaminase, oligouridylate bindin protein, Cullin,β-amyrin synthase, MADS -box protein, and starch branching enzyme. The results showed that only the positive yeast clone transformed with pGBKT-7-GmCASc and pGADT-7-MADS-box could grow on the SD/-Ade/-Leu/-Trp/-His plate and displayed the activity ofβ—gal. The above results showed that there was interaction between GmCASc and MADS-box. It was infered that the synergistic interaction of GmCASc and MADS-box in cell apoptosis and signal transduction might be one reason of soybean cytoplasmic-nuclear male sterility.