Cloning and Expression Analysis of Disease-related Gene SiRAR1 in Foxtail Millet (Setaria italica)
BAI Hui1, SONG Zhen-Jun1,2, SONG Dan-Dan1,3, WANG Yong-Fang1, QUAN Jian-Zhang1, DONG Zhi-Ping1,*, LI Zhi-Yong1,*
1 Institute of Millet Crops, Hebei Academy of Agriculture and Forestry Sciences/National Foxtail Millet Improvement Center/Minor Cereal Crops Laboratory of Hebei Province, Shijiazhuang 050035, China; 2 College of Life Sciences, Hebei Normal University, Shijiazhuang 050024, China; 3 College of Modern Science & Technology, Hebei Agricultural University, Baoding 071000, China
Abstract：Required for Mla12-required resistance (RAR1) as an important element for plant disease resistance, is widely involved in plant resistance response. To study the structure and expression characteristics of SiRAR1 in foxtail millet (Setaria italica), the CDS sequence and promoter sequence of SiRAR1 gene were separately cloned with the leaf cDNA and genomic DNA from resistance material 'Shilixiang' as template by PCR, and the biological characteristics of SiRAR1 were predicted by bioinformatics analysis. Then the expression patterns of SiRAR1 in different tissues and the Uromyces setariae-italicae-induced stress response were surveyed by qRT-PCR, respectively. Lastly the prokaryotic expression characteristics for the gene were detected by SDS-PAGE. The results showed that the ORF of SiRAR1 gene contained 765 bp and encoded 254 amino acids (GenBank No. MK814879). The predicted protein molecular weight was 28.04 kD and the theoretical isoelectric point (pI) was 8.13. The largest secondary structure element of SiRAR1 protein was random coil, and the smallest was beta turn. Phylogenetic analysis showed that SiRAR1 protein had the closest evolutionary relationship with plants in the same family, and had the highest genetic homology (92.44%) with the hypothetical protein C2845_PM12G15490 (RLM79685.1), a gene from chromosome 12 of Panicum miliaceum. The qRT-PCR analysis showed that SiRAR1 gene was expressed in roots, stems, leaves and panicles in foxtail millet. There was the highest expression level in roots and the lowest in stems, and the expression levels of SiRAR1 were similar in panicles at booting and heading stages. The qRT-PCR analysis also showed that under the infection of Uromyces setariae-italicae, the expression level of SiRAR1 gene in the resistant material 'Shilixiang' was up-regulated from 12 to 36 h and peaked at 36 h, and up-regulated at 96 h in the susceptible material 'Yugu1'. The peak expression of SiRAR1 gene in 'Shilixiang' was about 3 times higher than that in 'Yugu1' (P<0.01), and its peak expression in 'Yugu1' was about 1.6 times higher than that in 'Shilixiang' (P<0.05). The results showed that the expression of SiRAR1 gene had earlier up-regulated time, longer duration and stronger up-regulated range in resistant plants than that in susceptible plants. The prokaryotic expression vector pET30a-SiRAR1 was induced to express SiRAR1 fusion protein with 33 kD by 0.1 and 0.4 mmol/L isopropyl β-D-thiogalactoside (IPTG), respectively. The results provide a theoretical foundation for further study on the SiRAR1 function and its disease resistance mechanism in foxtail millet.
白辉, 宋振君, 宋丹丹, 王永芳, 全建章, 董志平, 李志勇. 谷子抗病相关基因SiRAR1的克隆及表达分析[J]. 农业生物技术学报, 2019, 27(12): 2091-2100.
BAI Hui, SONG Zhen-Jun, SONG Dan-Dan, WANG Yong-Fang, QUAN Jian-Zhang, DONG Zhi-Ping, LI Zhi-Yong. Cloning and Expression Analysis of Disease-related Gene SiRAR1 in Foxtail Millet (Setaria italica). 农业生物技术学报, 2019, 27(12): 2091-2100.
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