The Molecular Identification and Preliminary Study on Mutagenic Mechanisms of Lotus Mutants Induced by Ion Implantation
|Keywords||Baiyangdian red lotus Ion implantation Isozymes RAPD SRAP|
Ion implantation, as a new biophysically mutagenic technique, has shown a great potential for horticultural plant breeding. Fe+ Ion beam implantation has been applied to the seeds of Baiyangdian red lotus, we found there had great changes in lotus irradiated by ion beam through the culturing research of lotus mutants, such as patterns and colours of the flowers, this changes greatly improve the value of lotus which is known as the one of the top ten traditional garden ?owers and an important aquatic economic plant. however, there has no investigation been focused on the lotus mutants irradiated by ion beam at the molecular level, what’s more, the reports concerning the mutation molecular mechanism of ion implantation are few.In order to detect the lotus mutants at the molecular lever and further understanding the mechanism of mutagenesis by ion implantation, the differences between the mutants and the control were analyzed by isozyme electrophoresis, RAPD and SRAP techniques. The main results were as follows:1.The POD and SOD isozyme zymograms of Baiyangdian red lotus mutants irradiated by ion-beam and the control were analyzed by PAGE electrophoresis. The result showed that ion implantation could induce the changes of POD and SOD isozyme patterns of lotus mutants, and there had big differences among the different mutants.2. The random amplified polymorphic DNA (RAPD) was used, and then the bands of mutants and the control in the radiation-sensitive sites were cloned to be sequenced for comparing their DNA sequences. The results indicated that the total base mutation rate of mutants was 0.87%, and there was different in the six mutants. The types of base changes included base transition, transversion, deletion, and insertion. Among the 159 base changes detected, the frequency of single base substitutions (61.01%) was higher than that of base deletions and insertions (38.99%), and the frequency of base transitions (44.65%) was 2.7 times of that of the base transversions (16.35%). The transitions between C and T accounted for largest proportion, A→G transitions and A→T transversions were also present at high frequency. Adenine, thymine, guanine or cytosine could be replaced by any of other three bases, except that there was no C→G substitution. However, thymine and Adenine was more sensitive to the irradiation than other bases. In our study, we found many purine bases around the purine mutational sites, and many pyrimidine bases around the pyrimidine mutational sites.3. Sequence-related amplifid polymorphism (SRAP) was also used to amplifying the genetic DNA of six mutants and the control. The DNA was amplified with the optimized SRAP reaction condition, of the 121 primer pairs tested, 10 primers could amplify stable and remarkable specific bands, primer polymorphism is 8.26%; the amplified products were checked by PAGE electrophoresis, a total number of 215 bands were generated, among which 83 bands were polymorphic, and the percentage of polymorphic bands was 38.6%; there were different polymorphic band ratios among the six mutants; and then the specific bands of mutants and the control seperated by agarose gel were cloned to be sequenced for comparing their DNA sequences. The results indicated that the total base mutation rate of mutants was 0.95%, and there was different in the six mutants. The types of base changes included base transition, transversion, deletion, and insertion. The frequency of single base substitutions (58.22%) was higher than that of base deletions and insertions (41.78%), and the frequency of base transitions (45.21%) was 3.5 times of that of the base transversions (13.01%). Adenine, thymine, guanine or cytosine could be replaced by any of other three bases, Adenine (A) changes occurred 60 times (including 31 deletion and 5 insertions), guanine (G) changes occurred 39 times (including 15 deletions), Cytosine (C) changes occurred 27 times (including 6 deletions and 4 insertions) and thymine (T) changes occurred 19 times (no deletion or insertion). By comparison, Adenine (A) was more sensitive to irradiation than other three bases. we found many purine bases around the purine mutational sites, and many pyrimidine bases around the pyrimidine mutational sites.