内容已删除

你好，我也刚拿到这个系统，对他的应用还不了解。不知道这个系统是否适用于转录抑制验证，证明我的蛋白能够抑制目标启动子的活性。

For promoter activity assay, the MaLBD promoter region
was amplified by PCR using the specific primers listed in
Supplementary Table S1. The PCR product was inserted into
the pGreenII 0800-LUC double reporter vector (Hellens et al.
2005) at the HindIII and BamHI sites to fuse it with the Firefly
luciferase (LUC) reporter gene (MaLBDs pro-LUC); a Renilla
(REN) luciferase under the control of the 35S promoter at the
same vector was used as a positive control. The construct
CaMV35S-REN/MaLBDs pro-LUC was transformed into tobacco
BY-2 protoplasts by polyethylene glycol (PEG)
methods as described previously (Abel and Theologis 1994).
The promoter activity is indicated by the ratio of LUC to REN.

Transient Assay in Protoplast
A dual luciferase assay was performed in the transient assay.
For transactivation analysis of MaLBDs, the coding sequence
of MaLBDs without the stop codon was cloned into the
reconstructed GAL4DBD vector as effector. The double reporter
vector includes a GAL4-LUC and a internal control
REN driven by the 35S promoter. GAL4-LUC contains five
copies of GAL4-binding element and minimal TATA region
of the 35S promoter of CaMV, and these sequences are located
upstream of the LUC.
For the assay of the binding activity of MaLBDs to the
MaEXPs promoter, two pGreenII vectors, pGreenII 0800-
LUC reporter vector and pGreenII 0029 62-SK effector vector,
were used (Hellens et al. 2005). The MaEXPs promoter
was cloned into pGreen 0800 at the HindIII and BamHI sites
to fuse it with the LUC reporter gene (MaEXPs pro-LUC).
MaLBDs were cloned into the pGreenII 62-SK vector. The
pGreenII 0800-LUC vector carried a REN under the control of
the 35S promoter as a positive control，

我们刚构好启动子载体，还在做，不晚！

楼主，问一下，你的这个实验做完了吗，你把构建好的载体转化农杆菌了吗？我们转化一直转不进去