Nicotiana consists of 76 species and many of these species are believed to germinate from ascendant amny old ages ago chiefly due to cistron duplicate and sexual hybridisation at which the genome size and DNA sequences are different from one another. However, there is still deficiency of grounds on the development of Nicotiana species. An experiment was carried out to follow the development of Nicotiana from cistron duplicate by comparing the noncoding DNA 5 sizes of QPT cistron of N. syl, N. check, N. suav, MATF1, N. gla and N, afr extracted from the foliages of the works species utilizing quantitative analysis with UV spectrophotometer and Nanodrop and qualitative analysis with PCR and gel cataphoresis. The sexual hybridisation of N. syl and N. Tom was investigated by comparing the PCR merchandises of N. check and MATF1. It was found that there were two sets for most of the Nicotiana species in agarose gel and the DNA set sizes of N. check were similar to that of MAFT1. This indicated that cistron duplicate and hybridisation of current species to organize new species have occurred. Further research has to be carried out by look intoing the IGS of N. Tom and N. syl and with other noncoding DNAs of QPT cistron.
Every being on this planet diverged from a common ascendant many old ages ago due to development procedures. Harmonizing to Dittmar & A ; Liberles ( 2011 ) , development is a procedure at which the familial stuff inherited is changed over the clip from one coevals to the other which increases the familial diverseness. This may due to natural choice as proposed by Darwin, familial impetus, cistron flow, non-random coupling or mutant which alter the genome. Of all the factors doing familial development, mutant plays the major functions. In eucaryotes, most the cistrons mutated due to duplicate of the individual cistron or the whole genome of the hereditary species which consequences in the divergency of new species.
In this experiment, the evolutionary history of Nicotiana is traced. Nicotiana is a genus with 76 species of herbaceous works derived from Solanaceae household ( Knapp et al. , 2004 ) . The cultivation of Nicotiana covers the scope of America, Australia, South Africa and South Pacific. Harmonizing to Qu et Al. ( 2004 ) , Nicotiana has a large genome and most of the species have duplicated genome. Besides that, it was besides found that some Nicotiana species are formed by the sexual hybridisation of two current species to bring forth allotetraploid which are the N. check from hybridisation of N. syl and N. Tom in South America ( Matzke et al. , 2004 ) . The evolutionary history of Nicotiana from duplicate and hybridisation can be determined through the analysis of esential cistrons that encode for functional protein in prolonging life.
QPT is a catalytic enzyme which maps in the synthesis of alkaloid and NAD/NADP as it is required to change over 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 cistron structures show distinguishable differences. The coding part ( coding DNA ) the QPT cistron is extremely conserved as the amino acids sequences of QPT encoded by these two cistrons in N. check and N. gla are rather same ( Ryan et al. , 2012 ) . However, the noncoding DNAs are non so extremely conserved due to different 5 ‘ regulative parts of the DNA sequence. Therefore, by comparing the Deoxyribonucleic acid sequences and size of noncoding DNAs of the QPT cistron, the presence of NQTP1 and NQPT2 will demo the evolutionary history of modern Nicotiana species.
This experiment was carried out to follow the evolutionary history of Nicotiana by look intoing the noncoding DNA 5 size fluctuation of N. syl, N. check, N. suav, MATF1, N. afr and N. gla. This is to find the presence of other version of QPT in the genome of these few species. The Deoxyribonucleic acid sequences within the atomic genome of N. tabacum and the MATF1 were compared to analyze any alterations in sizes done to the Deoxyribonucleic acid of the hereditary parental species during natural hybridisation. Besides, this experiment was carried out to larn the methods in pull outing Deoxyribonucleic acid from cells and understand the rudimentss of recombinant DNA engineering.
Materials and methods:
In this experiment, the Deoxyribonucleic acid of each different works foliage tissues was extracted by first taking 75mg fresh weight of the tissue from healthy foliage tissue and was placed into a labelled micro-centrifuge tubing utilizing forceps. 75Aµl of CTAB extraction buffer was added utilizing micropipette and the works tissues was land to a all right paste. After that, 675Aµl of extraction buffer was added and the content was assorted exhaustively. 600Aµl of this works homogenate was transferred to a tubing incorporating 600Aµl of chloroform//isoamyl intoxicant ( 24:1 ) in the smoke goon and the content was assorted. The tubing was so placed in the warming block at 65EsC located in the fume goon with the palpebra unclosed. After 10 proceedingss, the tubing was removed and cooled to room temperature for 3 to 4 proceedingss. The content was assorted to make an emulsion. Centrifugation was so carried out for 10 proceedingss at full velocity to precipitate out the cellular dust and proteins. The upper DNA aqueous stage was removed and transferred to a new micro-centrifuge tubing. 600Aµl of isopropyl alcohol was added to the new micro-centrifuge tubing incorporating the DNA solution and assorted. The tubing was so centrifuged at full velocity for 15 proceedingss to pellet out the nucleic acid. After that, the supernatant of the tubing was discarded and the DNA pellet was added with 500Aµl of 70 % ethyl alcohol. It was assorted to take drosss and salts and was centrifuged for 5 proceedingss. With the 70 % ethyl alcohol removed, the pellet left was dried at room temperature 5 to 10 proceedingss and re-dissolved in 30Aµl distilled H2O. The process of DNA extraction was repeated for N. syl, N. check, N. suav, N. gla, N. afr and MATF1.
For each of the species, the solution was so run for gel cataphoresis by adding 5Aµl of the sample solution and 5Aµl of lading dye incorporating glycerin and bromophenol blue marker dye to the gel wells to find the quality of Deoxyribonucleic acid from the extraction. The optical density of DNA for each Nicotiana species was measured at 260nm and 280nm utilizing UV spectrophotometer and Nanodrop methods. In PCR reaction, 5Aµl of DNA sample for each Nicotiana species was added to micro-centrifuge tubings incorporating 5Aµl of oligonucleotide solution A with primer A, 5Aµl of oligonucleotide solution B with primer B and 20Aµl of distilled H2O. The tubings were subjected to PCR reaction with 30 Tens PCR cycling ‘s in thermocycler for 1.5 proceedingss at 92EsC, 1.5 proceedingss at 52EsC and 2.5minutes at 72EsC. After that, the tubings were maintained at 72EsC for 10 proceedingss to guarantee the PCR reaction was completed and were cooled to room temperature. The PCR merchandises were run for gel cataphoresis with a BSTEII-digested I» DNA which acted as DNA marker and the consequences were captured with UV light.
Consequences and treatment:
Table1. The optical density of DNA samples from different works species utilizing Nanodrop method
Ratio, OD260: OD280
Final nucleic acid concentration, ng/AµL
Initial nucleic acid concentration, ng/AµL
Deoxyribonucleic acid concentration, ng/AµL
Table2. The optical density of DNA samples from different works species utilizing UV spectrophotometer method
Ratio, OD260: OD280
Final nucleic acid concentration, ng/AµL
Initial nucleic acid concentration, ng/AµL
Concentration of DNA in the nucleic acid samples, ng/AµL
Table3. The figure of seeable DNA sets and the set sizes observed in PCR gel for different samples
Number of DNA sets
Deoxyribonucleic acid set sizes, kilobit
Quantitative analysis of Deoxyribonucleic acid samples
In this experiment, the DNA concentration was calculated utilizing the optical density of DNA measured with UV spectrophotometer and Nanodrop method. This was carried out to find the pureness of DNA extracted. Harmonizing to Beer Lambert jurisprudence, the optical density measured is straight relative to the concentration of the sample. This is because the optical density is the sum of light absorbed by the atoms ( Cantle, 1982 ) . When the figure of atoms in the sample additions, the optical density besides increases. The higher the figure of atoms in the sample the higher the concentration of the sample will be. Hence, by mensurating the optical density of the DNA, the concentration can be calculated.
The optical denseness of Deoxyribonucleic acid in the sample was measured at wavelength of 260nm and 280nm. As stated by Reece ( 2004 ) , DNA contains jumping individual and dual bond in DNA bases which are the A, G, C and T. Each of these nucleotide bases has different soaking up spectrum and the overall soaking up spectrum will be the norm of these four spectra. Therefore, DNA will demo a maximal soaking up at 260nm wavelength. The optical density of 280nm was measured to find the pureness of nucleic acid as taints such as proteins, saccharides and cell constituents will impact the optical density of nucleic acid at 260nm and therefore doing the consequence to be inaccurate. Harmonizing to Keer & A ; Birch ( 2008 ) , the ideal ratio of optical density at 260nm and 280nm is 1.8 to 1 as the Deoxyribonucleic acid is considered pure.
From the consequence, it was shown that the optical denseness ratio of nucleic acid samples for all the Nicotiana species measured utilizing UV spectrophotometer and N. tab utilizing Nanodrop at 260nm and 280nm was lower than that of the ideal scope. This may due to taint of proteins and saccharides. As stated by Alaey et Al. ( 2005 ) , the optical density of Deoxyribonucleic acid samples at 280nm will increase by the presence of aromatic amino acids. This will do the optical density ratio to be lower than the acceptable scope. Besides, the uncomplete remotion of cellular constituents such as cell dust will besides be one of the grounds. Besides that, the low optical denseness ratio may ensue from improper handling of the Deoxyribonucleic acid during the process of pull outing DNA which causes some of the solutions such as isopropyl alcohol and ethyl alcohol were non to the full removed from the infusions. As for the Nanodrop optical ratio, merely the samples of N. syl and N. suav were in the ideal scope. The remainder of the samples have ratio higher than the ideal scope. This may due to the presence of RNA in the samples as they are non separated from the Deoxyribonucleic acid.
Besides that, the concentration calculated from the optical density 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 mistakes during the dilution of the samples. By and large, it can be seen that Nanodrop is more efficient than UV spectrophotometer in mensurating optical density. This causes the spectrophotometer to be capable of mensurating Deoxyribonucleic acid with highly low concentration from 2 to 15000ng/AµL. As a consequence, there was non a demand to transport out dilutions for the Deoxyribonucleic acid samples to forestall out of scope. Finally, this can minimise any parallax mistakes which will take to inaccurate optical density measured. In contrast, UV spectrophotometer requires samples dilutions as it can merely mensurate narrow scope of concentrations ( Sambrook & A ; Russell, 2001 ) .
The extraction of Deoxyribonucleic acid can be improved to obtain better consequences by adding the RNase to divide RNA from DNA to mensurate the optical density. Further purification of the DNA infusions should be carried out.
Qualitative analysis of PCR merchandises
From the consequence, it can be seen that for the PCR gel image of N. check, MATF1, N. suav and N. gla, there were two discernible DNA sets which were 1.0kb and 0.8kb severally. During the PCR reaction, the oligonucleotide forward primer, NtQPTEx5 and change by reversal primer, NtQPTEx6 were used to magnify the noncoding DNA 5 sequence. Since there were two sets observed from the agarose gel cataphoresis of the PCR merchandises, it can be seen that there are two different version of the noncoding DNA 5 at which the sizes were different. This indicated that the QPT cistron is duplicated in Nicotiana species as NQPT1 and NQPT2 has low degree of preservation of the size and sequence of noncoding DNA 5.
However, there was merely one set observed for N. syl. This is because there were really two sets overlapping each other, doing merely one denser set to be noticed due to uncomplete separation as the sizes of the two sets were rather closed to each other. As for the N. afr, it was observed that there were three sets separated in the agarose gel. This may due to the ground that other than duplicate of QPT cistron, there was besides hybridisation of two bing species to organize N. afr. Hence, three sets were observed. Besides that, it was observed that the DNA sets in the agarose gel for N. check and MATF1 were similar in sizes which were 1.0kb and 0.8kb. Besides, one of the DNA sets of N. check was same to the DNA set of N. syl which was 1.0kb.This showed that hybridisation between N. syl and N. Tom resulted in the formation of new species which is the N. check as it has same QPT cistron as the MATF1 which was produced by sexually traversing of N. syl and N. Tom by Monash.
The noncoding DNA 5 sequence of NQPT1 and NQPT2 of N. gla were 1.0kb and 0.7kb severally harmonizing to NCBI genbank. This indicated that the upper DNA set on the gel was the noncoding DNA 5 of NQPT1 while the lower set with longer migration distance was the noncoding DNA 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 noncoding DNAs of N. gla bulk of them were longer than that of N. check while most of the noncoding DNAs of NQPT2 of N. gla were shorter than that of N. check.
In order to obtain more accurate consequence, the development of N. check can be traced by analysing the IGS of N. syl and N. Tom. Harmonizing to Volkov et Al. ( 1999 ) , IGS served as the familial markers which allowed the development of N. check rDNA to be traced. Similarities between the rDNA of N. check and its hereditary species, N. syl and N. Tom will uncover the evolutionary relationship. Besides, other noncoding DNAs of the Nicotiana species can be used for PCR and gel cataphoresis such as the noncoding DNA 2. This is because the lengths of noncoding DNA 2 of NQPT1 and NQPT2 for N. gla are mostly varied from that of N. check at which NQPT1 intron 2 of N. gla is much larger.
In decision, N. check was produced from the hybridisation of N. syl and N. Tom as the sizes of noncoding DNA 5 for NQPT1 and NQPT2 of N. check were similar to that of the MAFT1. Besides, one of the DNA sets of N. check was in the same size with that of N. syl. The presence of two DNA set on the agarose gel after gel cataphoresis of PCR merchandises indicated that there was duplicate of the QPT cistron in hereditary species which resulted in more than one version of QPT cistron. Hence, the hypothesis is accepted.