Analysis of transgenes
After Agrobacterium-mediated rice calli transformation, several hygromycin resistant rice lines were obtained, and their further molecular analysis were done. The genomic DNA of T0 transformants were extracted to do PCR amplifying HPT and Cre. Then PCR positive lines were selected to detect four foreign genes (PSY, CrtI, BHY and BKT). The PCR results of some lines showed the expected bands of four genes were detected (Figure 3).
Figure 3 PCR assays of several transgenic rice lines. M, Marker 2K plus. CK+, positive control (plasmid pYLTAC380MF-BBPC). WT, negative control (wild-type rice cultivar HG1).
To detect expression levels of these four foreign genes involved in astaxanthin biosynthesis, total RNA of transgenic rice seeds were extracted and their cDNA was synthesized from 1 μg of DNase-treated RNA. The results of RT-PCR showed the expected bands of the four key genes for synthesizing astaxanthin in transgenic rice seeds (Figure 4).
Figure 4 RT-PCR analysis of expression levels of PSY, CrtI, BKY, and BHY genes in several transgenic rice. Rice OsActin1 was as an internal control. CK+, positive control (plasmid pYLTAC380MF-BBPC). WT, negative control (wild-type rice cultivar HG1).
These results of PCR and RT-PCR demonstrated that we successfully obtained transgenic rice with all of four stacking genes (PSY, CrtI, BHY and BKT).
In summary, we successfully obtained transformants with all of transgenes in lab and detected obvious transcriptional activity in endosperm. These results indicated that our designed pathway was realizable in rice endosperm. To further confirm astaxanthin production, several analysis were carried out. The results were shown in Demonstrate page.