The miniature Ping (mPing) is a recently discovered endogenous miniature inverted repeat transposable element (MITE) in rice, which can be mobilized by tissue culture or irradiation. It is reported here that mPing, together with one of its putative transposase-encoding partners, Pong, was efficiently mobilized in somatic cells of intact rice plants of two distinct cultivars derived from germinating seeds subjected to high hydrostatic pressure, whereas the other autonomous element of mPing, Ping, remained static in the plants studied. mPing excision was detected in several plants of both cultivars in the treated generation (P0), which were selected based on their novel phenotypes. Southern blot analysis and transposon-display assay on selfed progenies (P1 generation) of two selected P0 plants, one from each of the cultivars, revealed polymorphic banding patterns consistent with mobilization of mPing and Pong. Various mPing excisions and de novo insertions, as detected by element-bracketing, locus-specific PCR assays, occurred in the different P1 plants of both cultivars. Pong excision at one locus for each cultivar was also detected by using a Pong internal primer together with locus-specific flanking primers in the P1 plants. In contrast to the pressurized plants, immobility of both mPing and Pong in control plants, and the absence of within-cultivar heterozygosity at the analysed loci were verified by Southern blotting and/or locus-assay. Sequencing at 18 mPing empty donor sites isolated from the pressurized plants indicated properties characteristic of the element excision. Sequence-based mapping of 10 identified mPing de novo insertions from P1 progenies of pressurized plants indicated that all were in unique or low-copy regions, conforming with the targeting propensity of mPing. No evidence for further mPing activity was detected in the P2 plants tested. In spite of the high activity of mPing and Pong in the pressurized plants, amplified fragment length polymorphism (AFLP) analysis denoted their general genomic stability, and several potentially active retrotransposons also remained largely immobile. Further investigation showed that the same hydrostatic pressure treatments also caused mobilization of mPing in the standard laboratory cultivar for japonica rice, Nipponbare. Thus, a simple and robust approach for in planta MITE-mobilization in rice has been established by using high hydrostatic pressure treatment, which may be useful as an alternative for gene-tagging in this important crop plant.
By using high-pressure treatment, two mutant lines were obtained from a genetically stable japonica rice cultivar Bijing38. Genomic DNA of the mutant lines, together with the original line (Bijing38), was either undigested or digested by Hpa IIMsp I, and then subjected to molecular analysis using two markers, ISSR and RAPD. Results indicated that changes in the PCR amplification profiles of both markers are apparent in the two mutant lines compared with the original rice cultivar, suggesting that there had been both sequence changes and DNA methylation modifications in the mutant lines. Southern blot analysis using diverse sequences, including two cellular genes (S2 and S3), a set of retrotransposons (Osr7, Osr36, Tos19 and more), and a MITE transposon family (mPing and Pong), confirmed the results, and indicated that changes in DNA methylation pattern, genomic structure, and possible activation of some transposons indeed occurred in the mutant lines. Moreover, these changes are stably maintained through selfed generations and in different organs. Thus, our results indicate that it is possible to obtain stable mutants in rice by high pressure treatments, and the molecular basis of the mutants may include both genetic and epigenetic changes. Therefore, high hydrostatic pressure seems a promising approach for plant mutagenesis.
The mutant strains of aspergillus oryzae (HP300a) are screened under 300 MPa for 20 min. Compared with the control strains, the screened mutant strains have unique properties such as genetic stability, rapid growth, lots of spores, and high protease activity. Random amplified polymorphic DNA (RAPD) and inter simple sequence repeats (ISSR) are used to analyze the DNA fingerprinting of HP300a and the control strains. There are 67.9% and 51.3% polymorphic bands obtained by these two markers, respectively, indicating significant genetic variations between HP300a and the control strains. In addition, comparison of HP300a and the control strains, the genetic distances of random sequence and simple sequence repeat of DNA are 0.51 and 0.34, respectively.
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