麻竹同源异型盒基因DlKNOX的克隆及表达分析

Cloning and Expression Analysis of DlKNOX Gene from Bamboo (Dendrocalamus latiflorus)

摘要:

为探讨同源异型盒（KNOX）基因在麻竹（Dendrocalamus latiflorus）茎秆发育中的作用，采用RT-PCR和RACE技术，从其幼茎中克隆了1个KNOX同源基因，命名为DlKNOX，其cDNA序列全长为1511 bp，包含5’UTR 196 bp、3’UTR 238 bp和编码区1077 bp。该基因编码含358氨基酸的蛋白，具有KNOX1、KNOX2、ELK和Homeobox KN等4个保守结构域，符合KNOX家族的特征，属于I类蛋白。生物信息学分析表明，该基因编码的蛋白与水稻OSH1的一致性最高（86%）。组织表达特异性分析表明，DlKNOX在节部的表达丰度最高，其次为幼茎，根中最低。DlKNOX基因在大肠杆菌（Escherichia coli）中经诱导表达，获得1条分子量约为82 kDa的重组蛋白，与预期的重组蛋白分子量一致（包含了MBP标签蛋白42.5 kDa和DlKNOX蛋白39.5 kDa）。该基因在大肠杆菌中的最适表达条件为28℃，0.3 mmol L^-1 IPTG诱导2 h。这为进一步研究DlKNOX在麻竹茎秆发育中的功能奠定了基础。

Abstract:

In order to reveal the function of homeobox (KNOX) gene in the stem development of Dendrocalamus latiflorus, a homologous gene of KNOX, named as DlKNOX, was cloned from the bamboo young stem using RT-PCR and RACE. The cDNA was 1511 bp in full length, including 5' UTR 196 bp, 3' UTR 238 bp and an ORF 1077 bp, which encoded a protein with 358 amino acids. The conserved domain analysis demonstrated that the protein contained KNOX1, KNOX2, ELK and Homeobox KN domains, which was in line with the characteristics of KNOX family, belonging to the class I protein. The bioinformatics analysis indicated that DlKNOX had the highest identities with OSH1 from rice for 86%. The expression analysis showed that DlKNOX expressed in knob with the highest level, next in stem, and the lowest in root. DlKNOX was expressed in Escherichia coli, and a recombinant protein about 82 kDa was obtained, which was consistent with the expected molecular weight of the recombinant protein composed of MBP (42.5 kDa) and DlKNOX (39.5 kDa). The optimum conditions for the expression of DlKNOX in E. coli were induced by 0.3 mmol L^-1 IPTG for 2 h at 28℃. These lay the foundation for further research on the function of DlKNOX in stem development of D. latiflorus.