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This experiment is to study evolutionary history of Nicotiana by using the variation in size of intron of the QPT gene. Various techniques are use in this experiment which is DNA extraction, gel electrophoresis, PCR, nanodrop and UV spectrometry. The objective of this experiment is to determine whether genome duplication occurred in ancestral species that give rise to modern Nicotiana. Gel electrophoresis help to determine the quality of DNA extracted. UV spectrometry and nanodrop are used to determine the quantity of the nucleic acids. PCR and gel electrophoresis are used to study the evolutionary history of Nicotiana which support that whether genome duplication occurred in ancestral species that give rise to modern Nicotiana. Besides, this experiment also supports that Nicotiana tabacum which is the commercial tobacco that produce through hybridization of Nicotiana sylvestris and Nicotiana tomentosiformis.
Tobacco plants are the common name for Nicotiana species in Nicotiana genus, which consist of about 70 species. The leaf of these plants is use to make tobacco product such as cigarette. Nicotiana is a native plant in Americas, Australia, some South Pacific islands and South West Africa. Nicotiana species have very large genome and there are some research that show that that ancestral gene duplication in Nicotiana genome in the early time of its evolutionary history (Ren & Timko, 2001). Besides, there is also evidence that some of Nicotiana species are alloploids which produce from hybridization of two ancestral species, lead to chromosome to double and produce fertile allotetraploid (Ryan et al., 2012). The example is N. tabacum which is the commercial tobacco that produce through hybridization of N. sylvestris and N. tomentosiformis that happened thousands years ago in South America.
Gene structures which are the exons and introns can be used to study evolutionary history. In this experiment, QPT genes were used where some Nicotiana species genome indicate the appearance of two different QPT structures which known as N-QPT1 and N-QPT2 (Sinclair et al., 2004). QPT genes produce QPT enzymes which is important in various reactions in living cells. In exons, based on the sequence and size, there is great conservation between QPT gene and Nicotiana species. The problem is in introns where low conservation between the QPT gene and Nicotiana species which depends on the DNA sequence and size. This is why intron was used in this experiment.
The aim of this experiment is to study evolution of tobacco. This experiment also performs to determine whether genome duplication occurred in ancestral species that give rise to modern Nicotiana. The other aim is to investigate whether N. tabacum resulted from the sexual hybridization of N. sylvestris and N. tomentosiformis.
Materials and methods:
Six types of plant materials were used which are leaf tissues from six species; Nicotiana sylvestris (N. syl), Nicotiana tabacum cultivar SC58 (N. tab), Monash artificial tobacco (MATF1), Nicotiana suaveolens (N. suav), Nicotiana glauca (N. gla) and Nicotiana Africana (N. afr). In the first week, DNA extraction, gel electrophoresis and PCR were done. DNA extraction process involved crushing leaf tissue, inverting tube, heating and centrifugation. Besides, various chemicals were used in DNA extraction process such as CTAB extraction buffer, choloroform, isopropanol and 70% ethanol. The DNA sample obtained was analyzed using gel electrophoresis in order to know whether reasonable proportion of high molecular weight DNA was obtained. Then, PCR was done to analyze the DNA extract. 15Î¼L PCR mix was provided in microcentrifuge and the following solutions were added in the following order; 5Î¼L crude DNA, 5Î¼L oligonucleotide solution A, 5Î¼L oligonucleotide solution B and 20Î¼L sterile water. The tube was mixed gently and the reaction was subjected to 30X PCR cycling as follows; 1.5min @ 92oC, 1.5min @ 52oC and 2.5min @ 72oC.
In week 2, quantifications of nucleic acids were done using UV spectrometry and nanodrop. In nanodrop, 1Î¼L of each sample was loaded and measured. In UV spectrometry, each sample was prepared in two ways which are 1Î¼L of DNA sample + 999Î¼L sterile water and 2Î¼L of DNA sample + 998Î¼L sterile water. The readings were measured at 220, 260, 280 and 320 nm. The PCR reaction done in week 1 was analyzed using agarose gel electrophoresis. A 40 mL, 1% agarose gel was prepared. The PCR reaction was mixed with Gel Red. The samples and the ladder were loaded and the gel was run. The picture of the gel with UV illumination was taken.
Results and discussion:
Qualitative/semi quantitative analysis of DNA by gel electrophoresis
DNA ladder MATF1 N.suav
Figure 1. Gel for analysis of DNA
The figure above show the sample DNA isolated and run in gel electrophoresis for analysis. Based on Figure 1, the quality of DNA sample isolated is not that good. The bands obtained for both samples show that the sample did contain genomic DNA. However, the bands also show that the samples also nuclear ribosomal rRNA. At the end of the band of both samples also show that the samples contained degraded DNA, RNA and short nucleotide (Refer appendix).
Quantification of nucleic acids using nanodrop and UV spectrometry
Table 1. The optical density at 260 nm and 280 nm, ratio of OD260 to OD280 and concentration (ng/Î¼L) of Sample 1 and 2 using nanodrop
Optical Density (OD)
Table 2. The optical density at 260 nm and 280 nm, ratio of OD260 to OD280 and concentration (Î¼g/Î¼L) of Sample 1 and 2 at different concentration using UV spectrometry
Optical Density (OD)
Sample calculation for the concentration of DNA in Sample 1 (MATF1):-
1 OD260 of double stranded DNA = 50 Î¼g/mL
A. Dilution factor = 1000
Concentration = OD260 Ã- dilution factor Ã- conc. of dsDNA for 1 OD260
= 0.001 Ã- 1000 Ã- 30 Ã- 50 Î¼g/mL
= 1500 Î¼g/mL ÷ 1000
= 1.50 Î¼g/Î¼L Ã- 50%
= 0.75 Î¼g/Î¼L
B. Dilution factor = 500
Concentration = OD260 Ã- dilution factor Ã- conc. of dsDNA for 1 OD260
= 0.008 Ã- 500 Ã- 30 Ã- 50 Î¼g/mL
= 6000 Î¼g/mL ÷ 1000
= 6.00 Î¼g/Î¼L Ã- 50%
= 3.00 Î¼g/Î¼L
*Same calculation was used for Sample 2
Based on Table 1, the ratio of OD260 to OD280 for both samples using nanodrop analysis are not good and cannot be accepted for further analysis as both ratios follow the optimum ratio stated in manual. The ratio of OD260 to OD280 which are 2.12:1 for sample 1 and 2.06:1 for sample 2 exceeds the optimum ratio which is 1.6 - 2:1.
Based on Table 2, all of the ratios obtained using UV spectrometry analysis cannot be accepted as they did not follow the optimum ratio OD260 to OD280, 1.6 - 2:1. The concentrations of the nucleic acids were calculated as shown in Table 2 and the sample calculation which show that the DNA obtained cannot be accepted for further analysis.
Analysis of PCR reaction by agarose gel electrophoresis
Figure 2. Gel for analysis of PCR reaction
The approximate sizes of the band were shown in Figure 2 and the size was determined by using Fermentas DNA ladder (refer to Figure 3 in appendix). Based on Figure 2, this show that the result supported the hypothesis that the QPT gene is duplicated in Nicotiana species and also supported that the duplication occurred in ancestral species which give rise to modern Nicotiana. This is because the figure shows that all of the species share two similar bands which show that all the species have the same gene of QPT. This shows that all of the species have both N-QPT1 and N-QPT2 genes. Some species have more than two bands which suggest that they are more modern species. Thus, trace of evolution pathway can be determine.
Based on Figure 2, both N. sylvestris and N. tabacum share one similar band which show that N. sylvestris is one of the ancestral parent of N. tabacum, which is the modern, commercial tobacco. This support the hypothesis that N. tabacum resulted from sexual hybridisation of N. sylvestris and N. tomentosiformis. MATF1 is F1 of hybrid between N. sylvestris and N. tomentosiformis, thus supposed MATF1 should have similar bands as N. tabacum. However, based on Figure 2, MATF1 and N. tabacum did share quite similar bands but changes in the size of DNA still happen which is due to inter-specific hybridisation.
The other way to test the hypothesis is by doing Southern blot or QPT phylogenetic analysis. Apart from intron 5, intron 7 can also be used in this study as it has big size variation and low % identity between the QPT genes.
In conclusion, genome duplication did happen in ancestral species of Nicotiana which lead to production of modern Nicotiana. Besides, the result also show that N. sylvestris is one of the ancestral parent of N. tabacum. When comparing MATF1 and N. tabacum which have the same parent, the result show that changes in the size DNA did happen due inter-specific hybridization
Ren, N. and Timko, M.P. (2001). AFLP analysis of genetic polymorphism and evolutionary relationships among cultivated and wild Nicotiana species. Genome 44: 559-571.
Ryan, S.M., Cane, K.A., DeBoer, K.D., Sinclair, S.J., Brimblecombe, R. and Hamill, J.D. (2012). Structure and expression of the qunolinate phosphorobosyltransferase. Plant Science 188-189: 102-110.
Sinclair, S.J., Johnson, R. and Hamill, J.D. (2004). Analaysis of wound-induced gene expression in Nicotiana species with contrasting alkaloid profile. Functional Plant Biology 31: 721-729.