| Rice is one of the most important food crops as well as a mode plant of grass. Study of rice gene function is great significance to rice genetic improvement and ensure national food security. Mutant characterization is one of the most straightforward and effective way to study gene function. Classic genetics studies usually exploit the spontaneous mutations, which occurred in an extremely low frequency can not meet the needs of scientific research. Therefore, creating mutants through manually introducing high frequency mutations into plants is essential for plant gene functional research. As one of the most widely used mutagen, T-DNA insertion can not only destroy the function of the insertion site genes, but also facilitate the tagged gene sequence isolation with its own sequence known. In addition, through adding different types of reporter systems into the T-DNA, it can also be applied to study the temporal- and spatial-profiles of the cis elements adjacent to the insertion site. In this study, a large rice T-DNA insertional mutant library and a small mutant library of mi RNA overexpression were established and provided a public technology platform to study gene functions in the genomic hierarchy. The following three results were obtained from the creating and utilizing large T-DNA insertional mutant library:1. The author participated in the transformation of 50,000 transgenic plants. To date, a mutant library with a total of more than 120,000 independent transformants has been established.2. A whole-life cycle phenotypical screening and recording was carried out for 3,000 T1 generation families. As a result, a total of 1,003 families showed mutation phenotype, representing a rate of 33.43%; the phenotypic data of mutant library were improved; a number of valuable research materials were obtained through insertion-phenotype primary co-segregation test. 3 co-segregation families were isolated from this group of materials: CZH0773(03Z11BC50), CZH1588(03Z11CA28), CZH1731(03Z11CE42).3. By genetic analysis of the 3 co-segregation families, the study found that in addition to the destruction effect of T-DNA insertion, the 35 S promoter in the T-DNA inducing gene enhancement and silence was also one of the causes of plant mutation. CZH0773 mutant was late heading, semi-dwarf, with slightly yellow leaves. T-DNA inserted into the 3’ end of the Os03g63050 gene. Mutation phenotypes of CZH1588 family were later heading and leaves were slightly yellow, but allelic mutant and experiments of overexpression and RNAi did not show mutation phenotype. In the short-day conditions, expressions of multiple heading related genes were significantly decreased in the mutant, while overexpression of Hd3 a in the mutant caused advance heading, simultaneously the mutant is GA-sensitive, indicating that the downstream pathways of Hd3 a and GA were not affected in the mutant. CZH1731 family showed dwarf, more tillers, and late heading. Although the above 3 mutants showed a strict co-segregation between phenotypes and T-DNA insertions, but genetic complementation experiments found that both complementary vectors and empty vector restored phenotypes of about half of the positive transgenic plants, suggesting that the genes destroyed by T-DNA insertion were not the causes of these mutations. The results stated that the mutant phenotypes were caused by the 35 S promoter in the T-DNA with the "enhancer" effect resulting in the enhance expression of gene near the insertion site. The results of 35 S promoter methylation sequencing indicated that phenotypes restored by complementary vectors transformation were due to 35 S promoter in the complementary vectors transferred once again arousing the 35 S promoter methylation, thereby inhibiting the 35 S promoter-driven gene expression. This inference can explain in addition to the regulation of T-DNA insertion on neighboring genes, also can explain the regulation of 35 S promoter on the other elements in T-DNA. Such as the hygromycin gene and GUS reporter gene in the T-DNA exhibited a high abundance of expression due to 35 S promoter driving, but after re-introduced vector containing 35 S promoter into the mutant, hygromycin resistance and GUS reporter gene expression were silent because of 35 S promoter methylation. In addition, a lower ratio of 35 S promoters could escape the regulation of methylation, still exhibited its "enhancer" function, and thus increased the complexity of the mutant phenotype analysis. This study revealed the 35 S promoter-induced multiple effects in T-DNA insertion mutants, and gives a reasonable explanation for some complex phenomena in T-DNA insertion mutants carrying the 35 S promoter, and also deepened the understanding of the mutant library.Another part of the research content was about creating a small mutant library of mi RNA overexpression and studying mi RNA genes functions. Based on the mutant phenotype of CZH1731 family was dew to mi R156 e overexpression, in order to systematically study the functions of mi RNAs in rice genome, 53 mi RNA genes were cloned from 62 mi RNA families and 52 genes got overexpression transformants. The main work: construction of overexpression vectors, genetic transformations, phenotypic identification and analysis, and genes functional verification and so on. The main results included:1. Some overexpression mi RNAs plants showed obvious phenotypes. mi R319-OX phenotype: the callus regeneration efficiency was only about 5% and the positive rate was about 50%, while the plant was short and easy to die. mi R529-OX showed more tiller and semi-dwarf. mi R167-OX phenotype: large angle tiller, absent apical dominance, and degradation of the panicle top. mi R160-OX also exhibited a large angle tiller. mi R399-OX phenotype: weak plant type, leaves end appeared withered and yellow due to phosphorus poisoning. High P stress showed P absorption efficiency of mutant was higher than wild type, simultaneously the mutant phenotypes of osa-mi R399 target genes also showed P poisoning.2. Study of rice mi R156 function. CZH1731 family screened from the mutant library showed dwarf, more tillers, late heading, and small panicles. The mutant phenotype was due to osa-mi R156 e overexpression caused by T-DNA insertion. Overexpression of osa-mi R156 e and osa-mi R156 e A phenocopied the mutant and showed a dosage effect of osa-mi R156 e. czh1731 M exhibited an obvious phenotype of absent apical dominance and impacted MAX/RMS/D pathway genes expression, suggesting that there may be a new pathway through osa-mi R156 e to controlling apical dominance and tillers. Ectopic expression osa-mi R156 e by GAL4-UAS system and Os TB1 promoter could significantly affect rice plant type and the experiments reversely illustrated moderately reducing the osa-mi R156 e expression could reduce tiller number but help to increase panicle size and yield per plant, and consistent with the ideal plant type for the purpose of improvement of rice plant architecture. |
PDF Full Text Request