关键词: 高温, MBF1c, 紫花苜蓿, 耐热性, 耐热相关基因

Abstract: High temperature negatively affects plant growth and development, and is one of the major abiotic stress factors limiting the growth of alfalfa (Medicago sativa) in southern China. The full-length coding sequence of the gene encoding Multi protein Bridging Factor 1c (MsMBF1c) was isolated from the alfalfa variety “Zhongmu 1”. The MsMBF1c protein in alfalfa was found to be homologous to AtMBF1c in Arabidopsis thaliana, with 72% similarity at the amino acid sequence level. The transcript levels of AtMBF1c were measured in the root, stem, leaf, flowers, and fruit; and changes in transcript levels were monitored under high temperature, drought, and the combination of these stress conditions. The highest transcript level of MsMBF1c was detected in the flowers, followed by the root, leaf, stem, and fruit. MsMBF1c was induced by high temperature, drought, and their combination (up-regulated by 4.21, 2.15, and 4.59 fold, respectively). The pBI121-35S:MsMBF1c overexpression vector was constructed and transformed into wild-type (WT) Arabidopsis seedlings. Overexpression (OE) lines without the separation of kanamycin resistance were obtained in the T3 generation. The OE lines were then crossed with the mbf1c mutant (MUT) to generate complementary lines (COM). The presence of the transgene in the plant materials was confirmed by PCR, and the transcript levels of AtMBF1c and MsMBF1c were determined by qRT-PCR. To evaluate the heat resistance conferred by MsMBF1c, the seed germination rates and seedling survival rates were determined for the OE, COM, MUT, and WT Arabidopsis lines. The seed germination rates differed slightly among the OE, COM, MUT, and WT lines (97.6%-100.0%) under normal conditions (P>0.05). After a heat stress treatment, the germination rate of WT decreased to 71.7%; that of the MUT line decreased to 66.0%, significantly lower than that of WT (P<0.05); and those of three individual OE lines and the COM line decreased to 79.3%-87.0%, significantly higher than that of WT (P<0.05). The seedling survival rate did not differ significantly among the OE, COM, MUT, and WT lines under normal conditions. However, after a heat stress treatment, the survival rate of WT decreased to 16.7%; that of MUT decreased to 10.0%, significantly lower than that of WT (P<0.05); and those of three individual OE lines and the COM line decreased to 40.0%-76.7%, which were significantly higher than that of WT (P<0.05). The expression levels of genes encoding key regulators, including HSFA1a, HSFA2, HSFA3, HSFB1, WRKY25, WRKY18 and DREB2a were analyzed in the OE, MUT, and WT lines by real-time PCR. Under normal conditions, the transcript levels of HSFA2, WRKY18, and DREB2a were low in the MUT line (0.33-0.47 of that in WT); while the transcript levels of HSFA2, HSFA3, HSFB1, WRKY25, WRKY18 and DREB2a in the OE lines were 1.74 to 3.80 fold their respective levels in WT. After heat stress, compared with WT, the MUT line showed significantly decreased transcript levels of HSFA2, HSFA3, HSFB1, WRKY18, and DREB2a, and the OE lines showed increased transcript levels of WRKY18 but none of the other tested genes. In conclusion, MsMBF1c is a functional conserved gene in the heat regulation pathway. Overexpression of MsMBF1c can complement the thermotolerance deficiency of the mbf1c mutant, and also enhance the thermotolerance of Arabidopsis at the seed germination and young seedling stages. The function of MsMBF1c may be similar to that of AtMBF1c, which regulates plant thermotolerance with other key heat resistance genes.

Key words: high temperature, MBF1c, alfalfa, thermotolerance, heat resistant gene