关键词:海岛棉; 抗黄萎病; 未知基因( GbVWR); 克隆; 基因表达 Cloning and Expression Analysis of a Functional Gene GbVWR Induced by Verticillium dahliae in Gossypium barbadense ZHANG Li-Jia**, ZHANG Yan**, RONG Wei, YANG Jun, ZHANG Gui-Yin, WU Li-Qiang, LI Zhi-Kun, WU Jin-Hua, MA Zhi-Ying, WANG Xing-Fen* North China Key Laboratory for Crop Germplasm Resources of Education Ministry / Key Laboratory for Crop Germplasm Resources of Hebei / Agricultural University of Hebei, Baoding 071001, China Fund:This study was supported by the Natural Science Foundation of Hebei Province (C2013204141), the Special Research Found for the Doctoral Program of Higher Education (20131302120002), and the Science and Technology Support Project of Hebei Province (14226308D). Abstract Verticillium dahliae is a destructive, soil-borne fungal pathogen that causes severe losses in cotton yield and fiber quality. Mining functional genes related to resistance against V. dahliae will benefit efforts to genetically improve crop plants. In this study, we identified a gene that involved in cotton defense against V. dahliaebased on screening the full-length cDNA library and suppression subtractive hybridization library (SSH) induced by V. dahliae in Gossypium barbadenseand Gossypium. hirsutum, respectively. Sequence analysis indicated that there was no any annotation in NCBI database, and we named the sequence from G. barbadense as GbVWR. We characterized GbVWR gene and analyzed its expression. The full length cDNA of GbVWR was 520 bp including a 198 bp open reading frame (ORF), encoding 65 amino acid residues. Bioinformatic analyses suggested that GbVWR belonged to secretory protein and tis theoretical isoelectric point was 5.32. Using pET-32a(+) as a fused expression vector, a recombinant plasmid pET32a-GbVWR was constructed. The recombinant protein was induced in Escherichia coliBL21 (DE3) with 1.0 mmol L-1 IPTG then GbVWR could express about 7 kD protein in E. coliBL21 (DE3). In addition, diverse cis-acting promoter elements involved in fungal elicitor response, hormone response, wound-response, and flavonoid biosynthetic gene regulation were discovered in the promoter region of GbVWR. qPCR analysis showed that expression level of GbVWR was the highest in roots, and was significantly induced by V. dahliae. Besides, GbVWR could also be induced by SA, ET, and GA treatments, respectively. In conclusion, GbVWR is a new functional gene, which involved in multiple signal pathways in cotton defense response to Verticillium wilt.
Keyword: Gossypium barbadense; Verticillium wilt resistance; GbVWR; Clone; Gene expression Show Figures Show Figures
图2 重组蛋白pET32a-GbVWR表达产物SDS-PAGE分析箭头所指为目的蛋白条带; M: 蛋白分子量标准; 1: pET32a(+)空载体37℃下诱导9 h; 2: BL21(DE3)空菌株37℃下诱导9 h; 3~4: pET32a-GbVWR 37℃下分别诱导9 h和24 h。Fig. 2 SDS-PAGE analysis of the expressed product of recombinant pET32a-GbVWRArrows indicate the target protein bands; M: protein molecular weight standard; 1: strains harbouring pET-32a(+) after induction for 9 hours at 37℃; 2: strains harbouring BL21 (DE3) after induction for 9 hours at 37℃; 3-4: total proteins of engineering bacteria strains after induction for 9 and 24 hours at 37℃, respectively.
图3GbVWR在海岛棉品系Pima 90-53不同组织中的RT-PCR分析同一处理时间内, “ A” 表示在0.01水平上存在极显著差异。Fig. 3 Tissue-specific expression analysis pattern of GbVWR by qRT-PCR in the G. barbadensePima 90-53“ A” means the significant difference at the 0.01 probability level at the same treatment.
图4GbVWR在棉花黄萎病菌胁迫下的RT-PCR分析同一处理时间内, “ A” 表示在0.01水平上存在极显著差异。Fig. 4 Expression analysis of GbVWRunder Verticillium dahliaestress“ A” means the significant difference at the 0.01 probability level at the same treatment.
图5 外源激素处理下GbVWR的表达模式分析同一处理时间内, “ A” 表示在0.01水平上存在极显著差异, “ a” 表示在0.05水平上存在显著差异。Fig. 5 Expression analysis of VWRafter treatment by hormones“ A” means the significant difference at the 0.01 probability level at the same treatment, “ a” means the significant difference at the 0.05 probability level.
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