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[摘要]
目的 克隆白桦Betula platyphylla一氧化氮合酶(nitric oxide synthase,NOS),探讨其生物信息及表达模式,并考察非生物胁迫对其NOS基因表达的影响.方法 通过RACE技术克隆了白桦NOS基因,命名为BpNOS.初步对该基因进行了生物信息学分析、分子进化分析、非生物胁迫处理以及信号诱导.结果 该基因全长1 806 bp,含有完整的开放读码框,编码601个氨基酸(Genebank登录号:KJ197336).BpNOS基因为不稳定疏水性蛋白,可能存在信号肽,具有跨膜能力,α-螺旋、延伸链、无规则卷曲分布于整个蛋白.分子进化分析结果表明,BpNOS基因与葡萄等物种的遗传距离较近,说明有着较近的亲缘关系;与大豆、苜蓿的遗传距离较远,说明其亲缘关系较远.BpNOS基因的表达具有节律性,在15:00时达到最大值,为0:00时的12倍.盐、低温、重金属镉胁迫均能抑制BpNOS基因的表达,但响应模式不同;水杨酸(SA)和外源一氧化氮可以促进BpNOS基因的表达.结论 非生物胁迫能够在一定时间范围内抑制NOS活性,SA和外源一氧化氮可以通过NOS途径调控内源一氧化氮的合成,为进一步研究白桦一氧化氮的代谢调控奠定了基础.
[Key word]
[Abstract]
Objective Nitric oxide synthase (NOS) is a kind of important NOS in organisms. The gene cloning and expression pattern analysis of NOS in plant laid the foundation for study on the biological functions of nitric oxide in plants. Methods NOS gene in B. platyphylla was cloned by RACE technology, named BpNOS. Bioinformatics and molecular evolution analysis, abiotic stress, and signal induced of the gene were preliminarily carried out. Results This full-length gene was 1 806 bp with the complete ORF, encoding 601 amino acids (Genebank ID: KJ197336). BpNOS was an unstable hydrophobic protein. This protein may exist signal peptide with transmembrane ability. The alpha helix, extension chain, random coil distributed throughout the protein. Molecular evolution analysis results showed that the genetic distance of BpNOS gene was closer to the gene genetic distance of grape species NOS, which indicates the relatively close relationship between them; While the genetic distance of BpNOS gene was farther from soybean and alfalfa, which explains that the relationship of them is far. The expression of BpNOS has rhythm, reached the maximum at 15:00, 12 times as at 0:00. The expression of BpNOS gene could be inhibited by salt, low temperature, and heavy metal cadmium stress, but in a different response pattern. The expression of BpNOS gene could be promoted by salicylic acid and exogenous NO. Conclusion Abiotic stress could inhibit the activity of NOS. The salicylic acid and exogenous NO could be synthesized by NOS pathway to regulate endogenous NO. This paper lays a solid foundation for the further research on the metabolic regulation of NO in B. platyphylla.
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[基金项目]
国家自然科学基金资助项目(31200463,J1210053);黑龙江省博士后启动基金资助(LBH-Q12166)