Sequence Analysis Of Thirteen Mutants Of Rice Induced By Maize DNA

Rice variation induced by exogenous DNA has become an important way to create new rice germplasms and varieties, but the underlying mechanism for mutagenesis remains rarely known. A number of rice mutants with a variety of phenotypic variation were successfully obtained by using a modified“Pollen-tube Pathway” to transfer maize genomic DNA fragments into rice receptor “Nipponbare”. 13mutants(V1~V13) with special phenotypic variation were selected as experimental materials in this study.The method of AFLP was used to analyze DNA polymorphism of the experimental materials. Besides,specific bands were sequenced and analyzed. This paper was designed to at the molecular level reveal the changes of the genomic DNA of the receptor caused by the introduction of exogenous DNA, and analyze these changes at the DNA level impact on the function of the gene, providing data for mutation by exogenous DNA introduction.First, 27 selective amplification primers were optimized from 64 pairs of AFLP primers, then 64 specific bands and 98 absent bands were obtained in the 13 rice mutants by using optimized primers. The results showed that transferring exogenous maize DNA into rice receptor could induce the change of DNA polymorphisms mapping of rice.The 64 specific bands were recovered to be cloned and sequenced, sequence analysis revealed that: 1)15185 bases were obtained from sequencing the 64 specific sequences, including 591 bases mutation. The total base mutation rate is 3.89%, the results indicated that transferring exogenous corn DNA into rice receptor can induce base mutations, and had high mutagenic effect. 2) Specific bands were resulted from base mutation of the restriction endonuclease recognition sequences or(and) the adjacent selective bases, or(and) DNA fragments insertion or deletion. The results indicated that the introduction of exogenous DNA could not only cause mutation of individual base in rice genome, but also the insertion or deletion of DNA fragments. 3) In all of the base mutation types, the frequency of base substitution(45.52%) was significantly higher than that of base insertion(28.44%) or deletion(25.04%), and base displacements mainly for single base substitution, base insertions and deletions in favor of occur multiple consecutive bases. It indicated that base substitution was the major mutation type. 4) In the 284 mutations of base, therewere 241 single base mutations, 18 double bases mutations, 4 continuous three bases mutations, 21 continuous multiple bases mutation, the frequency of single base mutation(84.86%) is significantly higher than that of double bases mutation(6.34%), continuous three bases mutation(1.41%) and continuous multiple bases mutation(7.39%), the results indicated that transferring exogenous corn DNA into rice receptor can induce base mutations, and the major base mutation type was single base mutation. 5) In base substitutions, the frequency of transition(65.80%) was significantly higher than that of transversion(34.20%). It indicated that base transition was the major mutation type of base substitution. 6) Through analysis bases on both adjacent sides of the mutation site, we found that the frequency of 16 kinds flanked base types descending order: ANT(9.86%) > CNG(8.80%) > ANG(8.45%) > CNT(7.75%) > TNT(7.39%)= CNA(7.39%) > ANA(7.04%) > GNC(6.34%) > TNG(5.99%) > CNC(4.93%) = TNC(4.93%) =TNA(4.93%) = GNT(4.93%) > GNA(4.23%) > GNG(3.52%) = ANC(3.52%), relatively, ANG, ANT, CNG prone to base mutation, GNG, ANC less prone to base mutation. 7) In 241 single base mutations, 31.54%of mutation(76 bases) was transitions and 65.15% of mutation(157 bases) was transversion. The frequency of inserting three bases was 1.24%, and the missing five bases accounted for 2.07%. The results suggested that base transition and transversion were the main mutation types in single base mutation, and the frequency of transition was significantly higher than that of transversion. 8) Among the 64 specific sequences, mutation of 18 specific sequences occurred in coding regions, including 20 synonymous and 23non-synonymous mutations. The results suggested that base mutation induced by exogenous DNA could lead to mutation of its coding amino acids. It could affect the structure and function of gene-encoded protein, which became the main reason that variants exhibited various traits.In this study, mutants induced by exogenous DNA were selected as experimental materials, used the molecular detection technology proved that biological mutagenesis is an effective way, preliminarily revealed the molecular mechanism of mutation by exogenous DNA introduction, providing data for biological mutagenesis for further research, it was very significant to provide the evidence for biological mutation, discover and take advantage of new rice genes.