Chen Si, Zeng Lei, Chen Shangwu, Sun Yangwu, Zhang Wen, Xu Haiying, Ma Huiqin
College of Agriculture and Biotechnology, China Agricultural University, Beijing 100193, China.
Sheng Wu Gong Cheng Xue Bao. 2010 Apr;26(4):530-7.
We induced embryogenic calli (EC) and non-embryogenic calli (NEC) from flower filaments of Vitis vinifera L. cv. Chardonnay about 10 days before full bloom. The callus were sub-cultured, observed and verified by somatic embryo induction. PCR primers for VvSUC12 and VvSCU27 were designed according to the corresponding sequences in GenBank. After RNA extraction with RNAplant for EC and NEC cell lines, we synthesized the 1st strand DNA for semi quantitative RT-PCR, and normalized the density of the bands against house-keeping gene Actin. The results of 31 cycles semi-quantitative RT-PCR showed that VvSUC12 was highly expressed in both EC and NEC, with higher expression intensity in NEC than in EC, but not reached the significant level; while the expression of VvSUC27 was only detected in EC, and the expression level was significantly lower than that of VvSUC12. We increased the semi-quantitative RT-PCR cycle number to 35 and found that VvSUC27 gene was weakly expressed in NEC, in EC the intensity of the band was increased comparing with 31 cycles, and the expression level was higher than that of NEC. The paper discussed the differential expression of the two sucrose transporters and their relationship with the sucrose in the tissue culture medium.
我们在霞多丽葡萄（Vitis vinifera L. cv. Chardonnay）盛花前约10天从其花丝诱导出胚性愈伤组织（EC）和非胚性愈伤组织（NEC）。将愈伤组织进行继代培养，并通过体细胞胚胎诱导进行观察和验证。根据GenBank中的相应序列设计了VvSUC12和VvSCU27的PCR引物。在用RNAplant提取EC和NEC细胞系的RNA后，我们合成了用于半定量RT-PCR的第一链DNA，并以持家基因肌动蛋白对条带密度进行标准化。31个循环的半定量RT-PCR结果表明，VvSUC12在EC和NEC中均高表达，在NEC中的表达强度高于EC，但未达到显著水平；而VvSUC27仅在EC中检测到表达，且表达水平显著低于VvSUC12。我们将半定量RT-PCR循环数增加到35，发现VvSUC27基因在NEC中弱表达，在EC中条带强度相对于31个循环有所增加，且表达水平高于NEC。本文讨论了这两种蔗糖转运蛋白的差异表达及其与组织培养基中蔗糖的关系。
