甘蓝型油菜BnWRKY15s基因克隆及锑胁迫下的表达分析
克隆甘蓝型油菜BnWRKY15s基因，探讨其在锑 (antimony，Sb)胁迫下的功能。
以甘蓝型油菜品种湘杂油512为材料，通过RT-PCR技术克隆BnWRKY15s基因，利用生物信息学工具和qRT-PCR分析其蛋白结构、理化性质、表达模式及在Sb胁迫下的表达。
成功克隆了BnA04.WRKY15和BnC04.WRKY15基因，其cDNA序列分别为960和996 bp，分别编码319和331个氨基酸。BnA04.WRKY15的氨基酸分子质量为34.63 ku，等电点为9.75；BnC04.WRKY15的氨基酸分子质量为35.81 ku，等电点为9.75。两者均为不稳定型亲水蛋白，主要二级结构为无规则卷曲，其次为α-螺旋。序列一致性分析表明：BnA04.WRKY15与白菜蛋白的相似性为99.06%，BnC04.WRKY15与甘蓝蛋白的相似性为99.09%。系统进化树分析显示：BnWRKY15s蛋白与白菜、甘蓝、花椰菜蛋白属于同一进化分支，亲缘关系较近。BnWRKY15s在根部的表达量最高，达到叶组织的99.1倍。经过75 mg/L Sb溶液处理0、1、3、6、12和24 h后，BnA04.WRKY15和BnC04.WRKY15的表达量呈先上升后下降再缓慢上升的变化趋势，且在叶组织中的表达量于处理1 h后达到峰值，而在根组织中的表达量于处理24 h后达到峰值。
在甘蓝型油菜中，BnWRKY15s基因可能作为关键转录因子参与调控Sb胁迫反应。
Cloning of BnWRKY15s Gene and Expression Analysis under Antimony Stress in Brassica napus
To clone the BnWRKY15s gene in Brassica napus and explore its role under antimony (Sb) stress.
Using B. napus variety Xianzayou512 as the material, the BnWRKY15s gene was cloned by RT-PCR. Bioinformatics and qRT-PCR were applied to analyze its protein structure, physicochemical properties, expression patterns, and its expression response to Sb stress.
The BnA04.WRKY15 and BnC04.WRKY15 genes were successfully cloned, with cDNA sequences of 960 and 996 bp, respectively; encoding 319 and 331 amino acids, respectively. The molecular weight of BnA04.WRKY15 was 34.63 ku, with an isoelectric point of 9.75; the molecular weight of BnC04.WRKY15 was 35.81 ku, with an isoelectric point of 9.75. Both proteins were identified as unstable hydrophilic proteins, with the dominant secondary structure being random coil, followed by alpha helix. Sequence alignment showed that BnA04.WRKY15 shared 99.06% similarity with B. rapa, while BnC04.WRKY15 had 99.09% similarity with B. oleracea. Phylogenetic tree analysis placed BnWRKY15s proteins in the same evolutionary branch with B. rapa, B. oleracea, and B. cretica, indicating a close evolutionary relationship. The expression level of BnWRKY15s was the highest in root, 99.1 times higher than that in leaf. After treatment with 75 mg/L Sb solution for 0, 1, 3, 6, 12, and 24 hours, the expression level of BnA04.WRKY15 and BnC04.WRKY15 initially increased, then decreased, followed by a slow rise. The highest relative expression level in leaves occurred at 1 hour, while the highest expression level in roots occurred at 24 hours.
BnWRKY15s may play a key role as a transcription factor in regulating Sb stress responses in B. napus.
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Keywords:
- Brassica napus /
- antimony stress /
- BnWRKY15s /
- gene cloning /
- transcription factor
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