Isolation Of A Helicase Gene From Medicago Sativa And Function Analysis In Abiotic Stress Tolerance

Alfalfa is one of the most important leguminous forage. However, the drought and saline soil exert bad impact on alfalfa yield with the deterioration of the ecological environment. Improving the abiotic stress tolerance of alfalfa is a basic strategy to increase alfalfa yield under adverse environment. So it is necessary to understand the abiotic stress tolerance mechanism of alfalfa for alfalfa breeding by genitic engineering methods. It was reported that the expression of some helicase genes could improve plant tolerance under salt stress. In this study, a helicase gene, named as MH1 (Medicago sativa helicase), was isolated from alfalfa by RT-PCR. Furthermore, the expression pattern and the function of MH1 were determined. The results and conclusions are as follows:Cloning and sequence analysis of MH1 geneAccording to the conserved motifs of helicases, we isolated a 1,221 bp cDNA clone from alfalfa by RT-PCR methord, and named it as MH1. The Genebank accession number is EF011022. The predicated coding region of MH1 encodes a protein of 406 amino acids which contains all the nine known conserved helicase motifs of the DEAD-box family and the typical motif of RNA helicases Walker motif A (A-X-X-X-X-G-K-T) was detected in its motif I. Phylogenetic tree, constructed according to DEADc region sequence of helicase proteins, revealed that MH1 protein was closer to translation initial factor 4Aâ…¢.MH1 localizated in nucleusTo determine the localization of MH1 protein in cell, we made the 35S::MH1-GFP construct. After onion epidermis transfection by gene gun bombardment, MH1-GFP fluorescence was detected mainly in nucleus. This result suggested that MH1 may play its role in gene regulation.MH1 expressed in vegetative organs of alfalfa and induced by abiotic stressesThe semi-quantitative RT-PCR analysis showed that MH1 was expressed in roots, stems and leaves of alfalfa. More important, the transcriptional level of MH1 was upregulated after alfalfa treated by mannitol, NaCl and H2O2.Expression of MH1 improved abiotic stress tolerance of Arabidopsis plantsTo understand the function of MH1 in stress tolerance, we transferred the MH1 gene driven by 35SCaMV promotor into Arabidopsis plants, and the Arabidopsis transformed with the vector only was used as control. After screened by antibiotic, thirty-six independent transgenic plants of T1 generation were identified. Further PCR and RT-PCR analysis indicated that MH1 was expressed in these transgenic lines. Transgenic seeds could germinate on medium containing mannitol, NaCl or MV, but the germination of the control seeds was retarded. The seedlings of transgenic Arabidopsis showed higher tolerance ability under drought, salt and oxidative stress conditions than the control plants. Furthermore, the transgenic plants grew better than the control ones under drought and salt condition.Expression of MH1 enhanced ROS scavenging and osmotic adjustment capacity of Arabidopsis plantsFurther analysis revealed that, after drought and salt treatment the MH1-expressed plants showed higher superoxide dismutase (SOD) and ascorbate peroxidase (APX) activities, higher osmotic adjustment capacity and proline content than the control plants. To understand the molecular mechanism of MH1 function, we determined the expression of some abiotic stress involved genes. The result revealed that, in MH1-expressed Arabidopsis plants, the expression of MSD1, P5CS1 and RD29A was increased.Our results suggested that MH1 involved in improving plant tolerance under drought, salt and the seedlings of transgenic Arabidopsis showed higher tolerance ability under drought, salt and oxidative stress conditions by enhanced the capacity in ROS scavenging and osmotic adjustment of cells. Since expression of MH1 could improve plant stress tolerance by regulating the expression of stress involved genes at several aspects, we suggest that MH1 gene can be used to improve crop stress tolerance by genetic engineering.