| 引用本文: | 韦莉,李霞,黄舒婷,ChanthaphooneKEOVONGKOD,
何龙飞,詹洁,王爱勤,肖冬.花生类受体蛋白激酶基因AhBAK1的克隆及原核表达[J].广西植物,2021,41(4):573-583.[点击复制] |
| WEI Li, LI Xia, HUANG Shuting, Chanthaphoone KEOVONGKOD,
HE Longfei, ZHAN Jie, WANG Aiqin, XIAO Dong.Cloning and prokaryotic expression of receptor-like protein kinase gene AhBAK1 in Arachis hypogaea[J].Guihaia,2021,41(4):573-583.[点击复制] |
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| 花生类受体蛋白激酶基因AhBAK1的克隆及原核表达 |
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韦莉1,李霞1,黄舒婷1,ChanthaphooneKEOVONGKOD1,
何龙飞1,2,3,詹洁1,2,3,王爱勤1,2,3,肖冬1,2,3*
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国家林业和草原局云南珍稀濒特森林植物保护和繁育重点实验室,昆明650201
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| 摘要: |
| BAK1是富含亮氨酸重复序列型类受体蛋白激酶,参与调控植物先天免疫反应过程中的程序性细胞死亡过程。实验室已有的花生铝胁迫转录组数据揭示AhBAK1为铝胁迫响应基因。为研究其在花生抗铝机制中的作用,该文分析了铝胁迫下AhBAK1在花生耐铝品种‘99-1507'和铝敏感品种‘中花2号'(‘ZH2')根尖中的转录变化,采用RT-PCR技术进行扩增AhBAK1的完整CDS序列,并对序列特征进行了分析。结果表明:AhBAK1显著响应铝处理,且在‘99-1507'中有着更强的诱导表达;AhBAK1包括625个氨基酸残基,属富亮氨酸重复区类受体激酶,具跨膜域和蛋白激酶催化结构域,不存在信号肽,预测定位于细胞质膜;我们进一步构建了含有AhBAK1激酶域的pGEX-6p-1-AhBAK1-CD重组质粒,体外诱导表达出约70kD可溶性蛋白,经凝胶亲和层析纯化,最终得到基于蛋白印迹实验(WesternBlot)验证正确的重组蛋白。为进一步研究AhBAK1的生物学功能和生化功能奠定了基础。 |
| 关键词: 花生,AhBAK1,基因克隆,原核表达,蛋白纯化 |
| DOI：10.11931/guihaia.gxzw201906029 |
| 分类号:Q943 |
| 文章编号:1000-3142(2021)04-0573-11 |
| 基金项目:国家自然科学基金(31701356);广西自然科学基金(2016GXNSFBA380223)[SupportedbytheNationalNaturalScienceFoundationofChina(31701356);GuangxiNaturalScienceFoundation(2016GXNSFBA380223)]。 |
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| Cloning and prokaryotic expression of receptor-like protein kinase gene AhBAK1 in Arachis hypogaea |
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WEI Li1, LI Xia1, HUANG Shuting1, Chanthaphoone KEOVONGKOD1,
HE Longfei1,2,3, ZHAN Jie1,2,3, WANG Aiqin1,2,3, XIAO Dong1,2,3*
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1. College of Agriculture, Guangxi University, Nanning 530004, China;2. Key Laboratory of Crop Cultivation and Tillage, Guangxi Colleges
and Universities, Nanning 530004, China;3. National Demonstration Center for Experimental Plant Science Education,
College of Agriculture, Guangxi University, Nanning 530004, China
1. College of Agriculture, Guangxi University, Nanning 530004, China; 2. Key Laboratory of Crop Cultivation and Tillage, Guangxi Colleges
and Universities, Nanning 530004, China; 3. National Demonstration Center for Experimental Plant Science Education,
College of Agriculture, Guangxi University, Nanning 530004, China
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| Abstract: |
| BAK1, an LRR receptor-like protein kinase, interacts with other receptor-like protein kinase and mediates the programmed cell death in plant innate immune response. Previous transcriptome analysis had revealed that AhBAK1 was responded to aluminum stress. In order to explore the role of AhBAK1 in peanuts under Al stress, the transcriptional changes of AhBAK1 between Al-tolerant cultivar ‘99-1507' and Al-sensitive cultivar ‘ZH2' under Al stress were analyzed. Furthermore, the full-length CDS of AhBAK1 was amplified by RT-PCR and analyzed. The results were as follows: AhBAK1 responded significantly to aluminum treatment, with much higher induced level in ‘99-1507'. Furthermore, the full-length ORF of AhBAK1 was cloned into pMD19T vector, encoding a protein with 625 amino acids and belonging to LRR receptor-like protein kinase family. It was predicted that AhBAK1 had transmembrane and protein kinase catalytic domains, and located in plasm membrane. The recombinant plasmid of pGEX-6p-1-AhBAK1-CD was further introduced into the Rosetta(DE3)to express a 70 kD soluble protein. The purified recombinant protein was verified by Western Blot analysis. The research set a foundation for further study on biological and biochemical functions of AhBAK1. |
| Key words: Arachis hypogaea, AhBAK1, cloning, prokaryotic expression, protein purification |
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