摘要
基于NCBI数据库中本氏烟(Nicotiana benthamiana)的烟草八氢番茄红素脱氢酶PDS基因(ABE99707)的核苷酸序列,设计并合成特异性引物,以烟草栽培品种红花大金元叶片总RNA为模板,通过PCR方法获得了烟草NtPDS基因的cDNA片段。序列分析表明,该基因编码区为1749 bp,编码582个氨基酸,推测该蛋白等电点为7.53,理论分子量为65.04 kD。通过构建融合表达载体pET-32a-NtPDS,并转化大肠杆菌BL21(DE3),在37℃下经1 mmol/L IPTG诱导4 h表达后,产生了以可溶性蛋白形式存在的NtPDS融合蛋白,并通过Western blotting验证融合蛋白获得表达。利用半定量RT-PCR技术进行组织表达模式分析发现,该基因在烟草的叶片、花和茎中均有表达,在根中没有表达。该结果为进一步研究烟草八氢番茄红素脱氢酶NtPDS的活性和生物学功能奠定了基础。
Based on the nucleotide sequence of phytoene desaturase gene of Nicotiana benthamiana published on NCBI,specific primers were designed and synthesized. With the total RNA from leaves of Nicotiana tabacum cv. Hongda used as the template,the NtPDS gene was obtained by PCR. Sequence analysis showed that the gene contained a full coding region of 1749 bp encoding 582 amino acids with the molecular weight of 65. 04 kD and the isoelectric point of 7. 53. The fragments of the NtPDS gene were cloned into the prokaryotic expression vector pET-32a and the fusion expression vector pET-32a-NtPDS was constructed,which was then transformed into Escherichia coli BL21（ DE3）.The fusion protein NtPDS in the form of soluble fraction was effectively expressed under 1 mmol/L IPTG concentrations at 37 ℃ for 4 hours. And then this result was proved by western blotting. The semi-quantitative PCR results revealed that the transcript of NtPDS gene was detectable in leaves,flowers,and stems,but no such transcript detected in the roots. It laid a foundation for the further study on the enzyme activity of NtPDS and its biological functions.
出处
《植物遗传资源学报》
CAS
CSCD
北大核心
2014年第4期838-843,共6页
Journal of Plant Genetic Resources
基金
中国烟草总公司基因组重大专项(110201201006)
贵州省科技厅农业攻关项目(黔科合NY字[2011]3047号)
中国烟草总公司重点项目(中烟办[2010]221号)
贵州省优秀青年科技人才培养对象专项资金(黔科合人字[2013]02号)
关键词
烟草
八氢番茄红素脱氢酶
原核表达
表达谱
Nicotiana tabacum
phytoene desaturase
prokaryotic expression
expression profile
