Nicotiana consists of 76 species and many of these species are believed to evolve from ancestor amny years ago mainly due to gene duplication and sexual hybridization at which the genome size and DNA sequences are different from one another. However, there is still lack of evidence on the evolution of Nicotiana species. An experiment was carried out to trace the evolution of Nicotiana from gene duplication by comparing the intron 5 sizes of QPT gene of N. syl, N. tab, N. suav, MATF1, N. gla and N, afr extracted from the leaves of the plant species using quantitative analysis with UV spectrophotometer and Nanodrop and qualitative analysis with PCR and gel electrophoresis. The sexual hybridization of N. syl and N. tom was investigated by comparing the PCR products of N. tab and MATF1. It was found that there were two bands for most of the Nicotiana species in agarose gel and the DNA band sizes of N. tab were similar to that of MAFT1. This indicated that gene duplication and hybridization of current species to form new species have occurred. Further research has to be carried out by investigating the IGS of N. tom and N. syl and with other introns of QPT gene.
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Every organism on this planet diverged from a common ancestor many years ago due to evolution processes. According to Dittmar & Liberles (2011), evolution is a process at which the genetic material inherited is changed over the time from one generation to the other which increases the genetic diversity. This may due to natural selection as proposed by Darwin, genetic drift, gene flow, non-random mating or mutation which alter the genome. Of all the factors causing genetic evolution, mutation plays the major roles. In eukaryotes, most the genes mutated due to duplication of the single gene or the whole genome of the ancestral species which results in the divergence of new species.
In this experiment, the evolutionary history of Nicotiana is traced. Nicotiana is a genus with 76 species of herbaceous plant derived from Solanaceae family (Knapp et al., 2004). The cultivation of Nicotiana covers the range of America, Australia, South Africa and South Pacific. According to Qu et al. (2004), Nicotiana has a big genome and most of the species have duplicated genome. Besides that, it was also found that some Nicotiana species are formed by the sexual hybridization of two current species to produce allotetraploid which are the N. tab from hybridization of N. syl and N. tom in South America (Matzke et al., 2004). The evolutionary history of Nicotiana from duplication and hybridization can be determined through the analysis of esential genes that encode for functional protein in sustaining life.
QPT is a catalytic enzyme which functions in the synthesis of alkaloid and NAD/NADP as it is required to convert quinolinic acid to nicotinic acid (Ryan et al., 2012). There are two different types of QPT which are the NQPT1 and NQPT2 at which the gene structures show distinct differences. The coding region (exon) the QPT gene is highly conserved as the amino acids sequences of QPT encoded by these two genes in N. tab and N. gla are quite same (Ryan et al., 2012). However, the introns are not so highly conserved due to different 5' regulatory regions of the DNA sequence. Thus, by comparing the DNA sequences and size of introns of the QPT gene, the presence of NQTP1 and NQPT2 will show the evolutionary history of modern Nicotiana species.
This experiment was carried out to trace the evolutionary history of Nicotiana by investigating the intron 5 size variation of N. syl, N. tab, N. suav, MATF1, N. afr and N. gla. This is to determine the presence of other version of QPT in the genome of these few species. The DNA sequences within the nuclear genome of N. tabacum and the MATF1 were compared to study any changes in sizes done to the DNA of the ancestral parental species during natural hybridization. Also, this experiment was carried out to learn the methods in extracting DNA from cells and understand the basics of recombinant DNA technology.
Materials and methods:
In this experiment, the DNA of each different plant leaf tissues was extracted by first removing 75mg fresh weight of the tissue from healthy leaf tissue and was placed into a labeled micro-centrifuge tube using forceps. 75Âµl of CTAB extraction buffer was added using micropipette and the plant tissues was ground to a fine paste. After that, 675Âµl of extraction buffer was added and the content was mixed thoroughly. 600Âµl of this plant homogenate was transferred to a tube containing 600Âµl of chloroform//isoamyl alcohol (24:1) in the fume hood and the content was mixed. The tube was then placed in the heating block at 65ËšC located in the fume hood with the lid unclosed. After 10 minutes, the tube was removed and cooled to room temperature for 3 to 4 minutes. The content was mixed to create an emulsion. Centrifugation was then carried out for 10 minutes at full speed to precipitate out the cellular debris and proteins. The upper DNA aqueous phase was removed and transferred to a new micro-centrifuge tube. 600Âµl of isopropanol was added to the new micro-centrifuge tube containing the DNA solution and mixed. The tube was then centrifuged at full speed for 15 minutes to pellet out the nucleic acid. After that, the supernatant of the tube was discarded and the DNA pellet was added with 500Âµl of 70% ethanol. It was mixed to remove impurities and salts and was centrifuged for 5 minutes. With the 70% ethanol removed, the pellet left was dried at room temperature 5 to 10 minutes and re-dissolved in 30Âµl distilled water. The procedure of DNA extraction was repeated for N. syl, N. tab, N. suav, N. gla, N. afr and MATF1.
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For each of the species, the solution was then run for gel electrophoresis by adding 5Âµl of the sample solution and 5Âµl of loading dye containing glycerol and bromophenol blue marker dye to the gel wells to determine the quality of DNA from the extraction. The absorbance of DNA for each Nicotiana species was measured at 260nm and 280nm using UV spectrophotometer and Nanodrop methods. In PCR reaction, 5Âµl of DNA sample for each Nicotiana species was added to micro-centrifuge tubes containing 5Âµl of oligonucleotide solution A with primer A, 5Âµl of oligonucleotide solution B with primer B and 20Âµl of distilled water. The tubes were subjected to PCR reaction with 30 X PCR cycling's in thermocycler for 1.5 minutes at 92ËšC, 1.5 minutes at 52ËšC and 2.5minutes at 72ËšC. After that, the tubes were maintained at 72ËšC for 10 minutes to ensure the PCR reaction was completed and were cooled to room temperature. The PCR products were run for gel electrophoresis with a BSTEII-digested Î» DNA which acted as DNA marker and the results were captured with UV illumination.
Results and discussion:
Table1. The absorbance of DNA samples from different plant species using Nanodrop method
Final nucleic acid concentration, ng/ÂµL
Initial nucleic acid concentration, ng/ÂµL
DNA concentration, ng/ÂµL
Table2. The absorbance of DNA samples from different plant species using UV spectrophotometer method
Final nucleic acid concentration, ng/ÂµL
Initial nucleic acid concentration, ng/ÂµL
Concentration of DNA in the nucleic acid samples, ng/ÂµL
Table3. The number of visible DNA bands and the band sizes observed in PCR gel for different samples
Number of DNA bands
DNA band sizes, kb
Quantitative analysis of DNA samples
In this experiment, the DNA concentration was calculated using the absorbance of DNA measured with UV spectrophotometer and Nanodrop method. This was carried out to determine the purity of DNA extracted. According to Beer Lambert law, the absorbance measured is directly proportional to the concentration of the sample. This is because the absorbance is the amount of light absorbed by the atoms (Cantle, 1982). When the number of atoms in the sample increases, the absorbance also increases. The higher the number of atoms in the sample the higher the concentration of the sample will be. Hence, by measuring the absorbance of the DNA, the concentration can be calculated.
The optical density of DNA in the sample was measured at wavelength of 260nm and 280nm. As stated by Reece (2004), DNA contains alternating single and double bond in DNA bases which are the adenine, guanine, cytosine and thymine. Each of these nucleotide bases has different absorption spectrum and the overall absorption spectrum will be the average of these four spectra. Therefore, DNA will show a maximum absorption at 260nm wavelength. The absorbance of 280nm was measured to determine the purity of nucleic acid as contaminations such as proteins, carbohydrates and cell components will affect the absorbance of nucleic acid at 260nm and thus causing the result to be inaccurate. According to Keer & Birch (2008), the ideal ratio of absorbance at 260nm and 280nm is 1.8 to 1 as the DNA is considered pure.
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From the result, it was shown that the optical density ratio of nucleic acid samples for all the Nicotiana species measured using UV spectrophotometer and N. tab using Nanodrop at 260nm and 280nm was lower than that of the ideal range. This may due to contamination of proteins and carbohydrates. As stated by Alaey et al. (2005), the absorbance of DNA samples at 280nm will increase by the presence of aromatic amino acids. This will cause the absorbance ratio to be lower than the acceptable range. Also, the incomplete removal of cellular components such as cell debris will also be one of the reasons. Besides that, the low optical density ratio may result from improper handling of the DNA during the procedure of extracting DNA which causes some of the solutions such as isopropanol and ethanol were not fully removed from the extracts. As for the Nanodrop optical ratio, only the samples of N. syl and N. suav were in the ideal range. The rest of the samples have ratio higher than the ideal range. This may due to the presence of RNA in the samples as they are not separated from the DNA.
Besides that, the concentration calculated from the absorbance measured for two different dilutions for each nucleic acid samples should be the same. However, the concentrations of DNA in different dilution of samples were different. This may due to parallax errors during the dilution of the samples. Generally, it can be seen that Nanodrop is more efficient than UV spectrophotometer in measuring absorbance. This causes the spectrophotometer to be capable of measuring DNA with extremely low concentration from 2 to 15000ng/ÂµL. As a result, there was not a need to carry out dilutions for the DNA samples to prevent out of range. Eventually, this can minimize any parallax errors which will lead to inaccurate absorbance measured. In contrast, UV spectrophotometer requires samples dilutions as it can only measure narrow range of concentrations (Sambrook & Russell, 2001).
The extraction of DNA can be improved to obtain better results by adding the RNase to separate RNA from DNA to measure the absorbance. Further purification of the DNA extracts should be carried out.
Qualitative analysis of PCR products
From the result, it can be seen that for the PCR gel image of N. tab, MATF1, N. suav and N. gla, there were two observable DNA bands which were 1.0kb and 0.8kb respectively. During the PCR reaction, the oligonucleotide forward primer, NtQPTEx5 and reverse primer, NtQPTEx6 were used to amplify the intron 5 sequence. Since there were two bands observed from the agarose gel electrophoresis of the PCR products, it can be seen that there are two different version of the intron 5 at which the sizes were different. This indicated that the QPT gene is duplicated in Nicotiana species as NQPT1 and NQPT2 has low level of conservation of the size and sequence of intron 5.
However, there was only one band observed for N. syl. This is because there were actually two bands overlapping each other, causing only one denser band to be noticed due to incomplete separation as the sizes of the two bands were quite closed to each other. As for the N. afr, it was observed that there were three bands separated in the agarose gel. This may due to the reason that other than duplication of QPT gene, there was also hybridization of two existing species to form N. afr. Hence, three bands were observed. Besides that, it was observed that the DNA bands in the agarose gel for N. tab and MATF1 were similar in sizes which were 1.0kb and 0.8kb. Also, one of the DNA bands of N. tab was same to the DNA band of N. syl which was 1.0kb.This showed that hybridization between N. syl and N. tom resulted in the formation of new species which is the N. tab as it has same QPT gene as the MATF1 which was produced by sexually crossing of N. syl and N. tom by Monash.
The intron 5 sequence of NQPT1 and NQPT2 of N. gla were 1.0kb and 0.7kb respectively according to NCBI genbank. This indicated that the upper DNA band on the gel was the intron 5 of NQPT1 while the lower band with longer migration distance was the intron 5 of NQPT2. This is because a larger fragment will migrate slower and has a shorter migration distance as compared to the smaller fragment. Besides that, for other NQPT1 introns of N. gla majority of them were longer than that of N. tab while most of the introns of NQPT2 of N. gla were shorter than that of N. tab.
In order to obtain more accurate result, the evolution of N. tab can be traced by analyzing the IGS of N. syl and N. tom. According to Volkov et al. (1999), IGS served as the genetic markers which allowed the evolution of N. tab rDNA to be traced. Similarities between the rDNA of N. tab and its ancestral species, N. syl and N. tom will reveal the evolutionary relationship. Also, other introns of the Nicotiana species can be used for PCR and gel electrophoresis such as the intron 2. This is because the lengths of intron 2 of NQPT1 and NQPT2 for N. gla are largely varied from that of N. tab at which NQPT1 intron 2 of N. gla is much larger.
In conclusion, N. tab was produced from the hybridization of N. syl and N. tom as the sizes of intron 5 for NQPT1 and NQPT2 of N. tab were similar to that of the MAFT1. Also, one of the DNA bands of N. tab was in the same size with that of N. syl. The presence of two DNA band on the agarose gel after gel electrophoresis of PCR products indicated that there was duplication of the QPT gene in ancestral species which resulted in more than one version of QPT gene. Hence, the hypothesis is accepted.