I am getting GUS expression in the anther of a plant but I am unable to confirm it by PCR by gene specific primer. Any suggestions?

I hope you are using softwares like Primer3, NetPrimer or GeneRunner for synthesizing your primers, Blast your designed primer and you would get to know if it will be targeting the desired gene or not. You can try this with your primers which are not working. If doesn't show the desired gene in BLAST result, so it will be something non-specific. Also check that Tm of your left/right primers are same. Chk these issues first. What is your PCR template? If its DNA, better try your primers with RNA.

If you have homologues of this gene in other taxa, try to align the sequences that are available and then see for the most conserved region. Design degenerate or specific primers from this conserved region, you should get good amplification.

Also if you are working with gene-GUS fusion constructs, then it would be rather simple to take one primer from your gene and other from the GUS region and then amplify. The only problem here would be the product size would be large.

Try out these options and then let me know, I can get back to u with other ideas.

All the Best

Regards

Mahalakshmi

Those plants must be sectorial chimeras generated from transformed germlines.

Expression patterns observed in transcriptional fusions can not always be reproduced using RT-PCR. There are many reasons for this, but the most common I guess is that you are not including in your promoter all of the elements that control expression of your gene in vivo. Are you obtaining this GUS signal in different transgenic lines? Because if observed only in one line it could be effect of the presence of other regulatory elements at the site of the insertion. Also, it is possible the promoter is OK, but the endogenous gene is subjected to postranscripcional regulation in anthers (maybe affecting RNA stability), so you can see GUS accumulation, but no RNA for the endogenous gene (these are only some possibilities that I can think of right now).

In order to confirm that your GUS fusion reflects the expression of your gene in vivo, I would suggest performing in situ hybridization and compare both expression patterns.

I am not sure if you have used a positive control and whether you got a PCR product from that. That could be a good indication whether or not your primers are working. If you have positive result from the control, one way to go is trying different amplification conditions putting the stringency level into consideration.

Good luck

Have you stained a negative control as well? I usually include 20% methanol in the buffer to inhibit the endogenous GUS activity in pollen. As for PCR, GUS is a very sensitive assay and I would try two rounds of PCR with nested primers to detect low abundance mRNA.

Good luck

Do you work with a transgenic line expressing the bacterial iudA gene? If yes, follow the instructions given by the other commentators. If not, you may experience unspecific GUS activity that we have described in tobacco anthers years ago.

ALWEN, A., BENITO MORENO, R.M., VICENTE, O., HEBERLE-BORS E. (1992) Plant endogenous ß-glucuronidase activity: how to avoid interference with the use of the E. coli GUS as a reporter gene in transgenic plants. Transgenic Research 1:63-70.

Gustavo Acevedo-Hernandez expressed what I want to say. Here I would like to add one more point. Gene splicing also can a reason for your failure. So it may help if you design more pairs of primers to check. Good luck

If your expression is weak or diffuse or irregular, you should consider the possibility that it may arise from a microbial contaminant, whose beta-glucuronidase may not be amplifiable with your PCR primers. The points others have made about positive and negative controls are also very important

Pollen can have strong nuclease activity associated with it. You need to do controls (PCR of genes of the plant with the same anther DNA extract).

There are a lot of good suggestions already including endogenous plant GUS activity and low RNA levels that will need nested PCR to reveal them. Another possibility is GUS remaining in cells after the RNA has gone - GUS is a stable protein and can survive for a long time after the RNA that encoded it has been degraded - it might even be passed on to daughter cells after a cell division, even though the daughter cells have not expressed the GUS gene themselves. That said, I think endogenous GUS seems the most likely explanation.

I support the answer by Richard. But if your expression is good in anther, it may be due to stable transformation and gus might have been integrated downstream of anther specific promoters in the genome.

For PCR amplication you may include both positive control and negative controls so that the PCR conditions could be validated and try to tune the PCR conditions for best amplification. For any stable transformation you should be able to get the amplification from DNA isolated from any tissue.

I agree with Erwin, pollen have a very high endogenous GUS

activity, you need to have a negative control. Do not

forget to fix anthers in the acetone before assay, this

will help. Also, you need to check your primers, RNA, cDNA

with positive control. In addition, you can also prove gene

expression in the anthers by immunolocalization.

It depends whether your gene is in the N or C terminal of GUS. Good framing will guarantee that your gene is expressed and therefore possible to amplify with gene specific primers. GUS expression is not enough to tell if your gene is there in the right frame except in the case your gene is between GUS and the promoter.

Good luck.

Can any body please upload the following paper which is talking about endogenous GUS expression in plants (tobacco)

"Plant endogenous ß-glucuronidase activity: how to avoid interference with the use of the E. coli GUS as a reporter gene in transgenic plants."?

Unfortunately, I don't have a reprint or copy left of the paper.

Taras was so kind to send me a copy of the paper. Here it is.

Thanks Erwin Sir, It would help me I feel...