河套蜜瓜花粉管通道法转化hALR基因

Transforming hALR Gene into Cucumis melo L. cv Hetao via Pollen-tube Pathway

为利用河套蜜瓜为生物反应器生产可用于治疗肝病的蛋白因子,以人胎肝mRNA为模板,经RT-PCR扩增获得了471 bp cDNA片段。克隆到pMD18-T载体,命名为pMD-ALR,测序分析结果与GenBank中报道的hALR编码序列完全相同。酶切回收hALR片段,插入到含有甜瓜果实黄瓜素(Cucumisin)基因启动子的果实特异性表达载体pPZP-CGN中,构建了hALR甜瓜果实特异性表达载体pPZP-CAN。花粉管通道法转化试验大田自花授粉花63朵,收获T0代果实27颗,结实率为42.9%。提取T0代种子无菌苗DNA,经PCR、PCR-southern和斑点杂交检测有13株呈阳性。

In order to use Cucumis melo L. cv Hetao as a bioreactor for production of human augmenter of liver regeneration （hALR） protein,471 bp cDNA hALR was amplified from the human fetal liver mRNA by RT-PCR with two artificially synthesized primers,then ligated into vector pMD18-T to construct pMD-ALR. The sequence analysis of pMD-ALR showed that the sequence was completely identical with that of bALK eDNA in GenBank. The fruit-specific expression vector pPZP-CAN was constructed by inserting bALK which was obtained from pMD-ALR by Hind Ⅲ and Sac Ⅰ restrictive digesting,into plant expression vector pPZP-CGN which contains Cncumis me/o L. cv cucumisin gene promoter. 63 hand-pollinating flowers were obtained via pollen-tube pathway transformation method in testing-farm,by which 27 fruits were achieved with 42.9 percentage of fruit-set. The DNA from To seedings were analyzed by PCR, PCR-southern and Dot boltting analysis and 13 positive were obtained.