桑树景天庚酮糖-1,7-二磷酸酶基因的克隆、原核表达与植物表达载体的构建

Cloning and Prokaryotic Expression of the Sedoheptulose-1,7-Bisphosphatase cDNA from Mulberry and Construction of Plant Expression Vector

景天庚酮糖-1,7-二磷酸酶(SBPase)是卡尔文循环过程中的关键酶。利用RACE技术得到景天庚酮糖-1,7-二磷酸酶基因全长cDNA,命名为MSBPase(GenBank登录号:DQ995346)。MSBPase全长为1527bp,该序列含有1个1179bp的完整开放读码框,编码393个氨基酸,蛋白质理论分子质量约为42.6ku,等电点为5.85,其氨基酸序列与其他植物中已分离的SBPase有很高的同源性。对MSBPase编码的蛋白质(命名为MSBPase)进行结构预测分析表明,该蛋白富含无规卷曲(coil),高达64.29%,其次是α-螺旋(helix),为22.19%,而β-折叠(strand)只有13.52%。将MSBPase编码区插入原核表达载体pET30a(+),并转化到大肠杆菌菌株BL21中,经过IPTG诱导,MSBPase融合蛋白在BL21菌株中成功表达。将得到的MSBPase编码区插入植物表达载体pBI121中,构建了MSBPase植物表达载体pBI121-SBP。

Sedoheptulose-1,7-bisphosphatase （SBPase） is a key enzyme in the regenerative phase of Calvin cycle. A full- length eDNA encoding SBPase （designated as MSBPase, GenBank accession No. DQ995346） was cloned from mulberry （ Morus alba var. rnulticaulis） by rapid amplification of eDNA ends （RACE）. The eDNA was 1 527 bp containing a 1 179 bp open reading frame which was deduced for encoding a peptide of 393 amino acids whose molecular mass was inferred to be 42.6 ku with its isoelectric point at 5.85. Sequence comparison analysis showed that the sedoheptulose-1,7-bisphosphatase from mulberry （MSBPase） had high homology to SBPases from other plants. It was prospected that the structure of MSBPase was rich in coils and helixes, and was poor in strands. The coding region of the MSBPase was inserted into an expression vector, pET30a （ ＋ ）, and transformed into Escherichia coli BL21. The fusion protein was successfully expressed with IPTG induction. The plant expression vector with this fragment under the control of 35S promoter was constructed. The gene cloned in this study may be an asset in the mulberry gene engineering and the results may be helpful to further study the regulation of SBPase.