Abstract: 【Objective】The aim of this study is to clone stress-induced protein kinase gene 1 (SIK1) gene, to analyze its molecular mechanisms and promote their applications in breeding. 【Method】 The total RNA from the leaves of alfalfa was used as the template to design the degenerate primers based on homology cloning strategy, and then the full-length ORF sequence of MsSIK1 was obtained through a combined reverse transcription-PCR (RT-PCR). Sequence analysis was performed by using homology comparision, the SMART website (http://smart.embl-heidelberg.de/) was used to simulate the protein structure. The subcellular localization transient expression vector was constructed and transformed into onion epidermal cell by particle gun. MsSIK1 and GFP were expressed in a fusion, which could be used to analyze the subcellular localization by the fluorescence signal. Real time-PCR was used to investigate the expression pattern under NaCl, ABA and drought stress, transgenic Arabidopsis plants was obtained by Agrobacterium. Expression identification of transgenic plants was performed by RT-PCR, after the transgenic plants were obtained, T₃-2, T₃-6, and T₃-10 transgenic lines were used for character identification of transgenic Arabidopsis under salt stress at seedling stage. Chlorophyll content and MDA content were investigated for functional verification of salt resistance between the wild type and T₃-2 ,T₃-6, T₃-10 transgenic lines.【Result】The results indicated that ORF of MsSIK1 was 2 478 bp and contained a single open reading frame of 825 amino acid residues. The protein C terminal has high homology with a variety of plant kinase, the simulation of protein structure indicated that the protein encodes a putative leucine-rich repeat receptor-like kinase with various domains. In the N-terminal region, a putative extracellular domain containing 10 amino acid leucine-rich repeats (LRR) was identified, and a transmembrane domain was identified. In the C-terminal cytoplasmic region, a serine/threonine protein kinase domain was predicted. The subcellular localization result suggested that MsSIK1 is located in the plasma membrane of onion epidermal cell. Real time-PCR indicated that the mRNA accumulation of MsSIK1 was induced by salt stress, abscisic acid (ABA) and drought. In the salt treatment, expression was induced quickly and the level of the product peaked at 4 h (about 7 times as to the control). In the drought treatment, MsSIK1 transcription was not induced immediately, but reached its maximum at 2 h (about 6 times compared with the control). In the ABA treatment, MsSIK1 transcripts accumulated rapidly and peaked after 3 h (about 6.8 times compared with the control). The RT-PCR identification of transgenic plants indicated that there were 6 lines with obviously band in T₁, and highest expression in T₁-10, but not detected in the wild type, therefore, it indicated that the foreign gene had been integrated into the chromosome of Arabidopsis thaliana and inherited in the offspring. Compared with the wild type plants, T₃-2, T₃-6, and T₃-10 transgenic lines grew well at seedling stage in salt treatment, indicating that MsSIK1 improved the salt tolerance in Arabidopsis thaliana. Additionally, compared with the control, the content of chlorophyll in transgenic Arabidopsis decreased less in NaCl treatment, among them, the chlorophyll content of wild type decreased by 77%, T₃-3 decreased by 53%, T₃-6 decreased by 44%, T₃-10 decreased by 35%; equally, the content of chlorophyll in transgenic Arabidopsis accumulated less in NaCl treatment, among them, the content of MDA in wild type was 1.3 times as that in T₃-10 line. 【Conclusion】MsSIK1 as a receptor like protein kinase was induced by a variety of stress and could increase salt tolerance by overexpression in A. thaliana.

Key words: alfalfa, MsSIK1, receptor like protein kinase, salt stress, functional study