| The mismatch repair (MMR) is a major pathway in DNA repair system,which is critical for maintaining genome stability and DNA replication fidelity,as it is responsible for the recognizing and repairing erroneous insertions, deletions and mismatch of bases newly arising during DNA replication and genetic recombination, as well, as during the repair of some forms of DNA damage. Rice is an important food crop and a prominent molecular model species for monocotyledonous plants. Some MMR genes have been annotated in Rice Annotation Project Database,one of which is OsMsh6 (Os09g24220), a homologue to Msh6 (At4G02070) in the MMR system of Arabidopsis thaliana. However, its function, diversity and the response of its insertion mutant to mutagenic treatment are still in a lack of understanding so far. Therefore, the diversity of rice OsMsh6 gene was analyzed and the mutagenesis of its insertion mutants induced by ion implantation was researched in the dissertation. The main results are as follows:1. The diversity of rice OsMsh6 gene. Equivalent amounts of genomic DNA isolated from Nipponbare (as the control) were pooled with that from other 192 rice varieties respectively for PCR to amplify the fragment from 2334 to 3433bp (function domain) in the OsMsh6 gene. After digestion of by CEL Ⅰ nuclease, the base mismatch was found in 24 of 192 heteroduplexes, accounting for 12.5% and suggesting the single nucleotide polymorphisms (SNP) compared with the Nipponbare. Sequencing results showed 2 SNPs in introns and 6 SNPs in exons. Based on SNP pattern, these 24 varieties could be divided into 4 types,2 varieties with 6 SNPs at position of 2867,2953,3051,3074,3093 and 3210bp in type 1,9 varieties with 1 SNP at position 3055bp in type Ⅱ,9 varieties with 1 SNP at position 3093bp in type Ⅲ and 4 varieties with 1 SNP at position 3093 and 3370bp in type Ⅳ, respectively. These results would be important to reveal the differences in radio-sensitivity and mutation frequency among different rice varieties.2. Molecular and phenotypic characterization of OsMsh6 gene insertion mutants. Three insertion mutants, NF9010, NF7784 and ND6011 with the Tos17 insertion at the 1st,8th and 3’-UTR, respectively, were characterized by triple-primer PCR and RT-PCR. The Tos17 insertion in OsMsh6 gene was confirmed and all 3 insertion mutants were homozygous. In both NF9010 and NF7784 mutant seedlings, partial OsMsh6 mRNA transcripts were detected with the primer sets that amplified upstream region of the insertion site, but it was not detectable with the primer sets at the downstream region or across the insertion site, indicating that NF9010 and NF7784 lack full-length, functional OsMsh6 mRNA despite the existence of truncated transcripts. However, all OsMsh6 transcripts were detected with primer sets that amplified the upstream, internal and downstream fragments of whole functional region in ND6011 mutant, but no transcript was detected with the primer set across the insertion site, suggesting that ND6011 has the functional mRNA, but not full-length mRNA. Agronomic traits, such as plant height, number of productive panicles, panicle length, seed-setting rate and mass of 1000 grains, of the insertion mutants was checked and compared with that of their wild type, Nipponbare. Results indicated that at least one trait for each mutant changed significantly. The mutant ND6011 just showed significant (P<0.05) decreased seed-setting rate, but both NF7784 and NF9010 displayed significantly (P<0.01) reduction in ear length and seed-setting rate, moreover plant height of NF9010 was also significantly lower (P<0.05). Therefore, some agronomic traits of Tosl7 insertion mutants of the OsMsh6 gene changed significantly, showing the mutator phenotype. Furthermore different insertion mutants displayed the different mutator phenotype, which is related to effects on OsMsh6 gene function of different Tos17 insertion sites. These findings provide foundation for investigating the function of the OsMsh6 gene in DNA repair and exploiting such mutants in induced mutation breeding in rice.3. Ion implantation mutagenesis of OsMsh6 gene insertion mutants. Ion beam implantation was carried out to mutant seeds by 1000,3000 and 5000 pulses with the influence of 2.6×1015 N+/cm for each pulse to the same sample. Test on physiological index of seedling and seed-setting rate of M1 plants in fields indicated that radio-sensitivity of insertion mutants increased, especially under higher dosage. RT-PCR analysis using the primer sets before Tos17 insertion showed that OsMsh6 expression level was unchanged in seedlings of mutant NF9010 and NF7784, but enhanced in both Nipponbare and mutant ND6011 seedlings possesing functional mRNA as the treatment dosage increased. Therefore it is inferred that OsMsh6 gene is involved in repair to DNA damage. In M2 population phenotypic mutations were observed, such as the mutations in leaf color and width, tiller number, plant height and fertility, and some mutants were confirmed by SSR. Phenotypic mutation frequency for Nipponbare, ND6011、NF7784 and NF9010 treated by different dosages was 0.54-1.01%,3.52-4.67%,3.39-7.58% and 2.81-14.2%, respectively. The mutation frequency of three mutants enhanced as the dosage increased and it was increased by 4 to 7 times above that of NB under same dosage. Mutations at DNA level was also detected in M2 plants from Nipponbare and NF9010 treated with a dosage of 3000 pulses of ion implantation.50 and 56 point mutations in 2 of 24 amplified fragments were detected in Nipponbare and NF9010 respectively. No insertion or deletion mutation was observed and only base substitutions were found, in which the frequency of base transition was higher than that of base transversion and the purine mutation was more than the pyrimidine. Frequency of point mutations in all mutants is higher than that in Nipponbare. These results are important to reveal the characteristic of mutagenesis induced by ion beam implantation and to construct the system for induced mutation breeding with higher efficiency using MMR mutants. |
PDF Full Text Request