花粉管通道法转化大豆的分子表征

Molecular Token of Transferred Soybean via Pollen-tube Pathway

采用花粉管通道技术将含有 gus基因的pBI12 1质粒DNA导入辽豆 14 ,从处理的 10 0朵花中获得 5 3粒种子。经PCR检测 5 3粒种子的幼苗 ,共检测出 6株阳性植株。采用RT PCR和GUS组织化学染色法对所获得阳性植株进行检测 ,其中 2株幼苗 (T0 代 17号和 33号 )均出现阳性结果 ,进一步对这 2株阳性植株进行Southernblot检测 ,同样获得了阳性结果。上述检测结果表明 ,gus基因已整合到染色体上并得到了表达。对转基因植株后代进行了PCR跟踪检测 ,结果从 17号株系的 2 8株中检测出 19株阳性植株 ,33号株系的 35株幼苗中检测出 17株阳性植株 ,表明 gus基因可以在转基因植株后代遗传。本研究结果为大豆花粉管通道转化技术提供了比较充分的分子生物学证据。

Plasmid pBI121 containing gus gene was transferred into soybean cultivars Liaodou 14 via pollen-tube pathway, 100 flowers were treated and 53 seeds were harvested. The plants germinated from the 53 seeds were detected by PCR and 6 plants showed positive results, But only 2 positive plants(called No.17 and No.33)were achieved by RT-PCR and histochemical GUS assay. The 2 positive plants were further determined by Southern -blot. These results showed gus gene had been integrated into chromosomes and expressed. The generations of the transgenic plants were also analyzed, 19 plants of 28 plants derived from No.17 plant line showed positive and 17 plants of 35 plants derived from No.33 plant line showed positive.It is showed that gus gene could be stably inherited. These results provided more sufficient molecular evidence to determine that transferring soybean can be operated via pollen-tube pathway.