Cloning And Resistance Analysis Of Peanut SOS Genes Family

Peanut (Arachis hypogaea L.) is one of the most important and widely planted croups in the word. Salt stress is an abiotics stress that affects the production and development of agriculture severely. More and more researches have paid close attention to salt injury and damage,focued on the breeding of salt tolerant plants and germplasm. Traditional breeding is slower and less precise because of the barriers between different species. However, gene engineering including transgenic technology have solved the problem. As we all know, SOS family involved in mediating the intracellular ion balance and plays an important role in the response of plants to salt damage and adaptation. In this article, AhSOS2, AhSOS3genes and the part of AhSOS1gene were cloned from peanut, On the basis of the analysis of their expression patterns, we study the functional of the part of genes by in vitro bingding analysis of transgenic rice under the salt. This paper is the first reported the peanuts in SOS gene family and its biological function. Main results were as follows:(1) Isolation and expression analysis of the peanut SOS family genesIn this study, SOS2and SOS3genes were isolated and harvested by RACE method from the peanut Luhua-14, and named as AhSOS2and AhSOS3. AhSOS2is a1462bp full-length cDNA, including a1341bp open reading frame, its coding protein AhSOS2has446amino acids, and is a member of serine/threonine protein kinases. AhSOS3is651bp open reading frame, which coding216amino acids, belongs to group of Ca2+binding protein. Quantitative real-time PCR (qRT-PCR) analysis showed that SOS family genes were expressed constitutively in peanut tissues, and could be induced to express under salt and drought treatments. When treated with250mmol L-1NaCl and30%PEG-6000, the gene expression level increased dramatically at different degrees. The results above indicated that the peanut SOS family genes may participate in and play roles under salt and drought stresses.(2) Vector construction and prokaryotic expression of AhSOS2and AhSOS3The coding region of AhSOS2and AhSOS3were amplified by PCR, and the recombined prokaryotic expression vectors of pET-28a-AhSOS2and pET-28a-AhSOS3were constructed successfully. By transforming the vector into E. coli strain of BL21(DE3). the corresponding bacteria transformants were harvested. The expression analysis indicated that band of51kDa and25kDa of fusion proteins could be induced seperately by1.0mmol L-1IPTG by SDS-PAGE electrophoresis, and the expression level improved gradually with the extension of induction time. And the degrees of induction could attain highest level after8h and6h induction. The results showed that not only AhSOS2but also AhSOS3could all be induced to express in prokaryotic expression system, and the expression levels could increase according to the induction time. These results would lay a foundation for the further study of physical and chemical properties of the AhSOS2and AhSOS3together with their corresponding protein products.(3) The in vitro and in vivo function investigation of peanut AhSOS2and AhSOS3In vitro, the spot assay and liquid culture assay of AhSOS2were carried out using its corresponding prokaryotic strains under different NaCl concentration treatment or treat time, and the results displays that the E. coli strain which contained recombinant plasmid could growth faster than the strain that only contained empty vector, and the colony growth state of AhSOS2growth better than those of empty vector after cultured for12h. The colony growth state of AhSOS3and its bacterial liquide concentration were different and better than those of bacterial stain that contained empty vector, and the differences attained to maximum after18h of cultivation. The results indicated that SOS family genes could improve the resistance of recipient bacterium to salt.The functional verification of AhSOS2showed that the ability of salt tolerance and adaptation was also changed and enhanced in AhSOS2overexpression rice. After treated by different concentrations of NaCl, the results showed that Na+concentration in AhSOS2transgenic plants is lower than that of control, and the variation trend of K+concentration is just opposite. And the peroxide resistance test also showed that, with the improvement of high salt concentration, the red-brown spots of control plants appeared significantly more than those of transgenic plants. These results indicated that AhSOS2may play a role in maintaining ion homeostasis in plants as well as resistance to oxidative stress.