Cloning and Expression Analysis of Disease-related Gene BcSGT1 in Non-heading Chinese Cabbage (Brassica rapa ssp. chinensis)
LIU Dong-Rang, HOU Xi-Lin, XIAO Dong*
State Key Laboratory of Crop Genetics and Germplasm Enhancement/Jiangsu Engineering and Technology Center for Modern Horticulture/Key Laboratory of Biology and Germplasm Enhancement of Horticultural Crops in Eastern China, Ministry of Agriculture, Nanjing Agricultural University, Nanjing 210095, China
Abstract：SGT1 (suppressor of the G2 allele of skp1) as an important element for plant disease resistance, is involved widely in plant cell cycling, stress response, protein ubiquitination and signal transduction processes, and it plays an important role in plant disease resistance. In order to study the structure and expression characteristics of the disease-related gene BcSGT1 in non-heading Chinese cabbage (Brassica rapa ssp. chinensis), the full-length cDNA sequence of BcSGT1 gene was cloned from the resistant variety 'Suzhouqing' by RACE technique. The expression analysis of gene was used by qRT-PCR. The expression pattern of Peronospora parasitica and Alternaria brassicicola induced treatment conditions, sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) technique was used to analyze the prokaryotic expression characteristics for the gene. The analysis results of sequence indicated that the full-length cDNA of BcSGT1 gene was 1 418 bp, and the open reading frame was 1 074 bp in length, encoding a total of 358 amino acids. The relative molecular weight was 39.77 kD, and the protein theoretical isoelectric point was 5.05 (GenBank No. AB495003). The evolutionary analysis of amino acid homologous system showed that the BcSGT1 gene of the non-heading Chinese cabbage had similar evolutionary relationship with the same family plant, and the highest homology (97%) was found with the chromosome 3 gene of Brassica rapa (Bra000741). The qRT-PCR analysis showed that under the infection of P. parasitica, the expression level of BcSGT1 gene in the resistant variety 'Suzhouqing' peaked at the 24 h, while the expression level in the susceptible variety 'Aijiaohuang' reached the peak at the 48 h. The peak expression of BcSGT1 gene in the resistant variety 'Suzhouqing' was about 2.1 times higher than that in the susceptible variety 'Aijiaohuang' (P<0.01). With the infection of A. brassicicola, the expression level of BcSGT1 in 'Suzhouqing' reached its peak at 12 h, while the expression level in the susceptible variety 'Aijiaohuang' peaked at 24 h, and the peak expression of BcSGT1 gene in the resistant variety 'Suzhouqing' was about 2.0 times higher than the peak expression in the susceptible variety 'Aijiaohuang' (P<0.01). The expression level of BcSGT1 in 'Suzhouqing' is significantly higher than the expression level in 'Aijiaohuang' after 24 and 48 hours of infection by P. parasitica, and 12 h, 24 h, 48 h infection by A. brassicicola (P<0.01). The prokaryotic expression vector was induced by isopropyl β-D-thiogalactoside (IPTG) to express a fusion protein with a relative molecular mass of about 39 kD. The BcSGT1 gene of non-heading Chinese cabbage played an important role in the infection of P. parasitica and A. brassicicola, and BcSGT1 successfully achieved fusion expression in Escherichia coli, which provided the conditions for further protein level research and transgenic function research. It will also provide an important theoretical value for the selection of high-yield, high-quality and disease-related non-heading Chinese cabbage varieties.
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