Study of mutation in tomato genomic DNA using low energy ion implantation or soybean DNA by RAPD

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1 Indian Journal of Biotechnology Vol 14, July 2015, pp Study of mutation in tomato genomic DNA using low energy ion implantation or soybean DNA by RAPD Hongying Duan, Yongang Yu, Jingyun Li, Wanshen Wang, Feifei Chen and Yanqing Zhou* College of Life Sciences, Henan Normal University, Henan Xinxiang , P R China Received 10 January 2014; revised 26 September 2014; accepted 10 November 2014 To analyze mutation in tomato genomic DNA by RAPD technology, certain factors influencing RAPD amplification, such as, DNA template, Taq DNA polymerase, Mg 2+, dntps and primer, were studied to optimize RAPD amplification reaction system. RAPD amplification of tomato genomic DNA after low energy ion implantation along with soybean DNA was successfully performed. Compared to the control, genomic DNA of tomato implanted with N + or Ar + ion beam displayed mutation. Further, total number of amplification band, number of band and rate of band increased when implantation dose of ion beam increased. The rate of band caused by Ar + ion beam was higher compared to that by N + ion beam under the same implantation dose. In addition, soybean DNA also had effect on tomato genomic DNA. In comparison to whole genomic DNA of soybean, the number and rate of band increased for random fractured genomic DNA of soybean, indicating that it might be easier for DNA fragment to enter into cell and integrate into genomic DNA of tomato as compared to the whole genomic DNA. Collectively, the rate of band was positively correlated with the implantation dose of ion beam, and the effect of soybean DNA fragment on tomato genomic DNA was higher compared to whole genomic DNA of soybean, which would provide basis for application of ion beam technology in tomato breeding. Keywords: Foreign DNA, ion beam, RAPD, tomato Introduction Tomato (Lycopersicon esculentum L.) is generally grown all around the world 1, and has high culinary and medicinal value 2. At present, tomato industry is facing expansion in terms of cultivation area and output. Tomato breeding has also achieved significant development, such as, disease resistance breeding, stress tolerance breeding, quality breeding, etc. However, it is necessary to breed further for new cultivars of tomato with distinctive characters in order to meet the requirement of market. In 1980s, it was realized that low energy ion beam can cause certain mutagenic effect when organism are implanted with it 3. Subsequently, many studies confirmed that low energy ion beam technology is an effective method of creating new genetic material because the radiation damage of ion beam on organism induces genetic variation 4. Compared to other breeding methods, low energy ion beam technology exhibits many *Author for correspondence: Tel: ; Fax: dxdhy@126.com, Luckyqing2004@126.com advantages, such as, low damage, high mutation rate, wide mutation spectrum and so on 5. Thus, it has novelty and practical value in genetic modification of organism. Low energy ion beam technology was first applied to rice by Yu et al in Later on, several important crop, such as, wheat, tobacco, cotton, soybean, rape and others, have successfully been bred 6. In addition, the etching and sputtering effects of ion beam on organisms would be very beneficial for foreign DNA to enter into cells 7, and so low energy ion beam biotechnology is simple and feasible to introduce foreign DNA to organisms as compared to transgenic technology However, research and application of low energy ion beam biotechnology in tomato is still in infancy. In the present study, effect of ion beam and foreign DNA on tomato genomic DNA was explored. The seeds of tomato were respectively irradiated by different doses of N + and Ar + ion beam, and then immersed into soybean DNA solution. These treated seeds of tomato were sown and allowed to grow. The variations in genetic material of tomato implanted with ion beam or introduced with soybean DNA were further analyzed by RAPD.

2 DUAN et al: STUDY OF MUTATION IN TOMATO GENOMIC DNA 365 Materials and Methods Plant Materials The seeds of tomato (Zhongza No. 9) were kindly provided by Vegetable Flower Institute of Agricultural Sciences, Beijing, P R China. The seeds of soybean (Zaoshu No. 2) were preserved in the laboratory. Culture of Soybean Seedling and DNA Isolation The seeds of soybean were sown in sandy soil after immersed into distilled water for 24 h and cultured at 25 C with a 14/10 h light/dark photoperiod of lux illumination intensity. Soybean seedlings with single-leaf were pulled out and mixed to extract genomic DNA by CTAB method. In the present study, DNA fragment of soybean genomic DNA was obtained by ultrasonication. Genomic DNA of soybean was randomly fractured by ultrasonic waves (power 400 W) at 450 pulses, giving 3 sec between each pulse, and then detected by agarose gel electrophoresis. Implantation of Ion Beam The implantation of ion beam was performed in Key Lab of Ionbeam Bioengineering, Zhengzhou University, Henan, P R China. The seeds of tomato were evenly put in TITAN ion implanter and respectively irradiated by N + or Ar + ion beam at 30 kev energy condition, and the irradiation doses of ion beam were 0, ions/cm 2 and ions/cm 2. Culture of Tomato Plant The seeds implanted with N + or Ar + ion beam (0, N + /cm 2, N + /cm 2, Ar + /cm 2 or Ar + /cm 2 ) were respectively immersed into 0.1 SSC buffer solution or 300 µg/ml DNA working solution, which consisted of soybean DNA and 0.1 SSC buffer solution, but seeds in control group (CK) were only immersed into sterile water; the soybean DNA in the present study was either whole DNA or random fractured DNA fragments of soybean genomic DNA. After immersion for 10, 24 or 48 h, seeds were separately took out and washed several times with sterile water and then seeds of tomato treated were sown in test field and grown under the greenhouse conditions with 20 C light and 10 C dark temperature cycle. After 7 d, tomato seeds germinated and seedlings with two leaves were transplanted in nutritive bowl one month later and allowed to grow. After two months growth, seedlings with five or six leaves were transplanted in the test field and allowed to grow at the above culture conditions. One month later, the leaves of tomato plants at strong seedling stage were clipped, and genomic DNA of tomato was extracted by CTAB method. RAPD Amplification In the present study, RAPD amplification was performed as follows: initial denaturalization at 94 C for 5 min, followed by 35 cycles of 94 C for 1 min, 36 C for 1 min and 72 C for 1.5 min, with a final extension cycle of 72 C for 8 min. In order to ensure the optimum reaction system of RAPD, some factors, such as, DNA template, Taq DNA polymerase, Mg 2+, dntps and primer, influencing RAPD amplification were separately analyzed in 25 μl reaction system. For DNA template (10, 20, 40, 60, 80, 100 & 120 ng), and Taq DNA polymerase (0.5, 1.0, 1.5, 2.0 & 3.0 U) different concentration gradients were tested. Similarly, concentrations of Mg 2+ (0.5, 1.0, 1.5, 2.0 & 2.5 mmol/l), dntps (0.10, 0.15, 0.20, 0.25, 0.30, 0.35, 0.40 & 0.45 mmol/l) and primer (0.05, 0.10, 0.15, 0.20, 0.25 & 0.30 μmol/l) were also optimized. In addition, 20 primers were selected out of 150 primers and listed in Table 1. Results Optimization of RAPD Amplification Reaction System Various factors influencing RAPD amplification, such as, DNA template, dntps, Taq DNA polymerase, primer and Mg 2+, were studied to optimize RAPD amplification reaction system (Fig. 1). In the study, distinct RAPD amplification were observed within the range of ng DNA template (Fig. 1a), μmol/l dntps (Fig. 1b), 1 U Taq DNA polymerase (Fig. 1c), 0.2 μmol/l primer (Fig. 1d) and mmol/l Mg 2+ (Fig. 1e) in 25 μl reaction system. Table 1 RAPD primers screened and used in the study Primer Sequence Primer Sequence S7 GGTGACGCAG S83 GAGCCCTCCA S22 TGCCGAGCTG S86 GTGCCTAACC S24 AATCGGGCTG S1318 GGCGCAACTG S31 CAATCGCCGT S1500 CTCCGCACAG S32 TCGGCGATAG S1516 CCTGCGACAG S62 GTGAGGCGTC S2080 AGGCGGCACA S65 GATGACCGCC S2084 CCCAAGCGAA S66 GAACGGACTC S2141 CCGACTCTGG S75 GACGGATCAG S2149 CTCTTCCGTC S78 TGAGTGGGTG S2155 GAGAACGCTG

3 366 INDIAN J BIOTECHNOL, JULY 2015 RAPD Analysis of Genomic DNA from Tomato Implanted with Ion Beam RAPD amplification of tomato genomic DNA was performed with 20 primers and the results are shown in Table 2. It is clear from the study that total number of amplification, number of band and rate of band increased in comparison to control, as well as when implantation dose of ion beam was increased. Compared to the control, the number of band was respectively 15 and 21, and rate of band was 12.90% and 16.41%, when implantation dose of N + ion beam was and N + /cm 2. Further, in case of Ar + ion beam, the number of band was respectively 18 and 24, and rate of band was 14.28% and 18.32%, when implanted with and Ar + /cm 2. Thus, the number of as well as rate of band caused by Ar + ion beam was found higher in comparison with N + ion beam. RAPD amplification results with certain primers are shown in Figs 2 and 3. In brief, genomic DNA of tomato implanted with N + or Ar + ion beam displayed mutational effects and rate of band was positively correlated with dose of ion beam. RAPD Analysis of Genomic DNA from Tomato with Foreign DNA Introduced by Ion Beam RAPD amplification results of genomic DNA of tomato treated with ion beam and soybean DNA are listed in Table 3. Compared to the control, total number of amplification band was 121 and 124 from Fig. 1 (a-e) Optimization results of RAPD amplification reaction system: (a) Effect of DNA template on RAPD amplification [M: DL2000, 1-7 was respectively amplification result with 10, 20, 40, 60, 80, 100 & 120 ng template DNA]; (b) Effect of dntps on RAPD amplification [M: DL2000, 1-8 was respectively amplification result with 0.10, 0.15, 0.20, 0.25, 0.30, 0.35, 0.40 & 0.45 mmol/l dntps]; (c) Effect of Taq DNA polymerase on RAPD amplification [M: DL2000, 1-5 was respectively amplification result with 0.5, 1.0, 1.5, 2.0 & 3.0 U Taq DNA polymerase]; (d) Effect of primer on RAPD amplification [M: DL2000, 1-6 was respectively amplification result with 0.05, 0.10, 0.15, 0.20, 0.25 & 0.30 μmol/l primer]; & (e) Effect of Mg 2+ on RAPD amplification [M: DL2000, 1-5 was respectively amplification result with 0.5, 1.0, 1.5, 2.0 & 2.5 mmol/l Mg 2+ ]. Table 2 RAPD amplification of tomato implanted with ion beam Treatment condition Total no. of amplification No. of Rate of (%) 0 (Control) N + /cm N + /cm Ar + /cm Ar + /cm Fig. 2 (a & b) RAPD amplification of tomato genomic DNA with primer S86 (a) and S22 (b). [M: DM2000; 1: Control; 2: with N + /cm 2 ; 3: Amplification of genomic DNA from tomato implanted with N + /cm 2 ; 4, 6 and 8: with N + /cm 2 and then respectively immersed into soybean full length DNA solution for 10, 24 & 48 h; 5, 7 and 9: with N + /cm 2 and then respectively immersed into soybean full length DNA solution for 10, 24 & 48 h; 10, 12 and 14: with N + /cm 2 and then respectively immersed into soybean DNA fragment solution for 10, 24 & 48 h; 11, 13 and 15: with N + /cm 2 and then respectively immersed into soybean DNA fragment solution for 10, 24 & 48 h.]

4 DUAN et al: STUDY OF MUTATION IN TOMATO GENOMIC DNA 367 Fig. 3 RAPD amplification of tomato genomic DNA with primers S1318 (a) and S65 (b). [M: DL2000; 1: Control; 2: with Ar + /cm 2 ; 3: Amplification of genomic DNA from tomato implanted with Ar + /cm 2 ; 4, 6 and 8: with Ar + /cm 2 and then respectively immersed into soybean full length DNA solution for 10, 24 & 48 h; 5, 7 and 9: with Ar + /cm 2 and then respectively immersed into soybean full length DNA solution for 10, 24 & 48 h; 10, 12 and 14: with Ar + /cm 2 and then respectively immersed into soybean DNA fragment solution for 10, 24 & 48 h; 11, 13 and 15: with Ar + /cm 2 and then respectively immersed into soybean DNA fragment solution for 10, 24 & 48 h.] Table 3 RAPD amplification of tomato with soybean DNA introduced by ion beam Treatment condition Total no. of amplification No. of Rate of (%) Control* *Control: 0 beam treatment; 1: N + ion beam and full length DNA of soybean genomic DNA; 2: N + ion beam and random fractured fragment of soybean genomic DNA; 3: Ar + ion beam and full length soybean genomic DNA; 4: Ar + ion beam and random fractured fragment of soybean genomic DNA tomato treated with N + or Ar + ion beam and whole genomic DNA of soybean, number of band was 17 or 18, and rate of band was 14.05% or 14.51%. When foreign DNA was randomfractured genomic DNA of soybean, total number of amplification band, number of band and rate of band were increased. Further, under different doses of Ar + ion beam, the number and rate of band were variable, while the number and rate of band showed little change along with different doses of N + ion beam. RAPD amplification results with some primers are shown in Figs. 2 and 3. Discussion RAPD technology is actually PCR amplification and these RAPD amplifications can be used to identify organisms 11,12. The variation in genomic DNA can also be detected because the mutations, such as, insertion, deletion, rearrangement and others, would lead to changes in binding site between primer and DNA template. Further, RAPD amplification can be influenced by DNA template, dntps, Taq DNA polymerase and others 13,14. In the present study, the concentration of DNA template, Mg 2+, dntps, primer and Taq DNA polymerase were all optimized, and the optimal reaction system of RAPD amplification was established. It is well known that implantation of ion beam could cause prominent biological effect on organisms, because the etching and sputtering effects of ion beam on cells would make chromosome aberration and then some genetic variations would occur 15,16. In the present study, RAPD amplification of tomato implanted with N + or Ar + ion beam exhibited increase compared to the control, and the number and rate of band increased when dose of ion beam increased. Similar effect was also observed in other studies 17,18. Perhaps, base mutation caused by ion beam reduced the hybridization efficiency between DNA template and primers, as a result band of RAPD amplification would emerge. In addition, the repairable damage on some chromosomes caused by ion beam could facilitate integration of foreign DNA into genomic DNA 19,20, and foreign DNA could lead to significant variation in genomic DNA 21,22. In the present study, N + or Ar + ion beam-mediated transfer of soybean DNA had evident effects on tomato genomic DNA. Further, in comparison with whole genomic DNA of soybean, the number and rate of band were higher when foreign DNA was random fractured genomic DNA of soybean, indicating that DNA fragment might be easier to enter into cell and would integrate into genomic DNA of tomato. In conclusion, the effect of ion beam on tomato genomic DNA was positively correlated with implantation dose, and rate of DNA aberration caused by Ar + ion beam was higher compared to that by N + ion

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