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以长春花激素自养型细胞株C20hi及其母株C20D为材料,构建抑制性差减cDNA文库,斑点杂交筛选后对阳性克隆进行测序,共得到104个不同的差异表达基因片段。将这些序列在GenBank中进行BLAST比对分析,其中28个与已知基因具有很高的同源性。对部分基因的分析发现长春花细胞C20hi中g10h-like、CrPS(Catharanthus roseus protein S)、HyPRP(hybrid prolin-rich protein)、PNAE(polyneuridine aldehyde ester-ase)-like等基因的差异表达可能与该细胞株生物碱超量合成有关;而细胞色素P45071D和CR5基因的差异表达则可能与其激素自养有关。此外,培养基中2,4-D的缺失引起的TDC等酶活性的增强也可能是C20hi细胞生物碱超量合成及激素自养的原因之一。
Suppression subtractive cDNA library was constructed by using the Pichia pastoris autotrophic cell line C20hi and its parent strain C20D. The positive clones were screened by dot blot hybridization, and 104 different differentially expressed genes were obtained. BLAST analysis of these sequences in GenBank showed that 28 of them were highly homologous with known genes. Analysis of some genes revealed that the differential expression of the genes g10h-like, Catharanthus roseus protein Cr (CrPS), hybrid pro-proline protein (HyPRP), polyneuridine aldehyde ester-ase Cell alkaloid over-synthesis; and cytochrome P45071D and CR5 gene expression may be related to their hormone autotrophic. In addition, the increase of enzymatic activity such as TDC caused by the loss of 2,4-D in the medium may also be one of the reasons for the over-synthesis of C20hi cell alkaloids and the hormone autotrophy.