I agree with Ali Mahmoudpour. Same class of functional genes doesn't mean that their gene sequences are always 'identical'. Are you sure that their gene sequences are identical (you mentioned 'their nucleotides are the same')? Their gene sequences may have conserved domains and variable regions.

Another things is:

If the DNA sequences of the 5 alleles in the 5 different loci are similar, you should design primers from their most variable regions. So, PCR amplification can specifically target the allele you want. In this case, primers should be unique to the allele on chromosome 3. You won't get a full-length CDS if the specific primers are designed within the gene.

Dear Ali Mahmoudpour  and Yuan-Yeu Yau

First of all thank you for your response and time. I think i can explain bit more here. We have done comparative transcriptomics in banana plants for identification of possible gene candidates for drought tolerance and selected based on their gene expression pattern......we have information like loci,chromosome and sequences etc...I would like to go for over expression of one such candidate gene in banana plants through Agrobacterium mediated transformation. Now my task is to have to amplify full CDS for this purpose. Our transcriptome study showed that a transcript sequence from chromosome 3, annotated as eg. Peroxidase 3... Once we mapped this gene to banana chromosome (Peroxidase 3, not considering nt variation) , we found it present in more than one chromosome (I am sorry earlier i mentioned nucleotides are same). 

Now what i need is

1.A full CDS of this gene of chr 3 need to be amplified. As Yuan suggested i cant go for internal primer (based on sequence variation) i could end of with truncated CDS.

or is it ok to amplify from genomic DNA (i mean with introns and also have additional nt sequences). So i have more freedom in primer designing

2. Moreover, i suspect chromosome localization role in gene expression pattern? So i am confused either i just go for Peroxidase 3 without worrying its origination or should give important to chromosome loci?. So i would like have expert opinion who might have experience on this regards, before proceeding into the experiment. 

As Ali suggested whether i have look into factors like......quantitative effect...cis and trans acting elements....etc. I want to have experts opinion on this?

Should gene selection be chromosome specific for over expression studies in plant system? - ResearchGate. Available from: https://www.researchgate.net/post/Should_gene_selection_be_chromosome_specific_for_over_expression_studies_in_plant_system#585c45ba404854688

Yes it is good information Are you collaborating with Dr Leela.your colleague of IIHR in this work,Good  luck.

 Hi Muthusamy Muthusamy,

1. Seems like that your project is on the right track. Peroxidase (POD) is known involving in stress tolerance, including drought tolerance (see attachment, 2016) which you are after. [http://aob.oxfordjournals.org/content/118/2/259]

2. Peroxidase has different isoforms (ie. isozymes). This is well known and have been reported (see attachment, 2016). Most likely the 5 alleles in the different 5 loci you mapped are POD isozymes (you mentioned they are genes of same class, and Peroxidase on Chromosome #3). Different POD isozyme genes can express at different plant developmental stages in different tissues/ organs. Different POD isoforms also response to different stresses. Their gene products have different biological roles.

3. If the 5 alleles in your finding are indeed POD isozymes, their DNA sequences can be different (some with minor difference). Since your transcriptome data pointed out that the specific peroxidase on the Chromosome 3 is the major peroxidase 'isozyme' responding to drought experiments, you naturally have to after this one for downstream experiments. In other words, you need to specifically PCR out this allele, instead of the other 4 alleles at different loci.

4. So, for you to fish out this allele by PCR, probably you need to do DNA sequence alignment among these 5 alleles (you said you know their chromosome number, the locus and their sequence data). And design a specific pair of primers to PCR out the Chromosome #3 allele. As Ali has already mentioned that this part the technical can be changeling, but there should always have tools to make it accomplish.

5. People have used cDNA or gDNA form for gene overexpression. Both are reported. However, usually gDNA size is (much) longer than cDNA form; therefore, in theory, for cloning purpose, cDNA (no introns) are easier.

Hi Ali,

Thanks.

1. You mentioned the selection marker gene in your previous post. The selection marker gene (SMG) can be readily removed through the aid of other systems (such as site-specific recombination system, ex. Cre-lox) or transformation strategies (ex. co-transformation of two binary vectors). [see attached paper]

2. The most challenging step for Muthusamy now is how to PCR out this unique allele (on chromosome #3) if it has very similar sequence as those of other 4 alleles. Especially, when they have only a few bases difference. For the worst situation, he might need to synthesize the whole sequence of the allele-of-interest for downstream experiments (if it is easier this way).

Hi Ali,

It good to meet you here too.

@  Muthusamy Muthusamy,

Very good examples describing different Peroxidase (POD) isoforms is in the attached paper (page 1762, including Fig.5 and Fig. 6). Look like both 'gel mobility' and their 'pI values' are different for those isoforms. From these results, it indicates that the sequences of those Peroxidase isoforms should not be identical.

Paper Title: Senescence- and drought-related changes in peroxidase and superoxide dismutase isoforms in leaves of Ramonda serbica

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Grab from the page:

"Separation of POD isoforms based on molecular weights showed differences in POD profiles between mature, young, and old leaves (Fig. 5). A form of high mobility that was present in young leaves disappeared in mature and old leaves, while a form of low mobility appeared only in old leaves.

If the relative water content (RWC) reduced to 50% and 20%, two additional isoforms with higher mobility than in mature leaves.

Separation of POD isoforms by their pI values revealed two anionic isoforms with similar pI (4.5) and two cationic ones with pI of about 9.3–9.0 (Fig. 6).

During ageing, anionic isoforms increased by 180% in mature and 280% in senescent leaves compared with young leaves, while drought had no influence on the amount of anionic isoforms.

On the other hand, the intensity of cationic isoforms was 4-fold higher in the leaves of 50% RWC, and two new isoforms with pI 8.4 and 8.6 appeared. In the leaves with 20% RWC, two cationic isoforms (pI 9.3–9.0) were 3-fold intensified compared with turgid leaves."

Hi Ali,

That is a good thought. However, people are already doing that. The third generation of biofuel are biofuel generated from microalgae. Microalgae become a biofinery and convert CO2 into carbohydrates (sugars) and lipids (TGA) using sunlight energy. The carbohydrates and lipids can be harvested and further processed into biofuels. To grow microalgae, it needs inputs such as CO2, certain nutrients and lights. For this, people/companies have already build microalgae ponds/containers near the industrial sites, and collect the CO2 (inject into the ponds/containers) directly from the emission generated by factories. This is one stone for two birds-- to reduce the greenhouse gas and to produce biofuel.

But there is one thing to be concerned: CO2 emission from factories can mix with other pollutants and toxic gases. How to obtain good quality of CO2 for microalgae to grow from the mixed gases becomes important.

See this short movie from University of Kentucky (USA) for microalgae to capture CO2 from a coal power plant. http://reveal.uky.edu/algae_part1_howitworks

@  Muthusamy Muthusamy,

Attached is another paper for reference. It shows that different Peroxidase isoforms (isozymes) response to different environmental challenge (stress).

Thanks Dr Yuan and Ali for your answers and also for your discussion on another interesting research topic..Bio fuel production. Dr.Yuan i ll go through the papers