| Rice is natived to tropical Asia and widely cultivated in China, gradually spread to all over the world. Increasingly attracting scientists from all over the world on the study of rice breeding, the breeding practices indicate that need to increase crop yield, can be summed up two effective means.One is form improvement, the second is heterosis utilization. The Pure of form improvement, they potential is limited; Heterosis is not combined with a modified form, the effect is generally poor.The related Breeding approaches and technologies,including high-tech, genetic engineering, will eventually be implemented to the excellent shape and strong heterosis, and it will contribute to increase production. However the content and distribution of auxin and cytokinin have an important influence on plant morphogenesis.As one of the major natural active form of auxin, indole-3-acetic acid (IAA) is able to be biosynthesized through two pathways which are dependent or independent on tryptophan. In the tryptophan biosynthetic pathway,excessive OsYUCCA-1expression increase the content of IAA in rice plants,showing excessive auxin phenotype whereas expression of down-regulated expression of rice plants which do not exhibit auxin sensitive abnormal phenotype thus affecting the growth of rice. In this paper, using RNA interference technology with endogenous rice osa-MIR528precursor as the backbone to design amiRNA. After the level of regulation of gene transcription and suppress the expression of these specific genes without changing the genetic background of rice. To study based on the influence of these genes in rice development,so as to improve plant genes, enhanced resistance to improve crop yields and lay a theoretical foundation. Main results obtained are listed as follows:(1) Based on the published sequences of OsYUCCA-1and IPT on the NCBI website,primers were designed to amplify corresponding target fragments. The gene of OsYUCCA-1and IPT specific amiRNA expression vectors pmiR528-OX3, pmiR528-OX6, pmiR528-OX8, pmiR528-T-3,pmiR528-T-5and pmiR528-T-10were constructed based on one-step PCR and MAGIC;(2) The amiRNA expression vectors was transformed into rice callus by Agrobacterium tumefaciens-mediated rice transformation. After Hygromycin resistance selection and calli differentiation, their respective transgenic plants were obtained.(3) Further optimization of tissue culture and gene transduction efficiency, For establishing a highly efficient and stable rice tissue culture regeneration system and hygromycin gene was amplified by PCR fragment regenerated plants were positive identification, the results confirmed the different fragments of RNA interference has been integrated into the receptor rice genome. we obtained two positive RNAi transgenic lines of pmiR528-OX3,three positive RNAi transgenic lines of pmiR528-OX8, two positive RNAi transgenic lines of pmiR528-T-3, seven positive RNAi transgenic lines of pmiR528-T-10, respectively;(4) Positive transgenic plants in fluorescent quantitative PCR detection results show that the transgenic rice OsYUCCA-1gene transcription level was reduced by30%～50%;This study obtained the corresponding function loss or reduce rice mutants, for the study of RNAi interference of variant and IAA content changes, parsing the function of the gene in rice response to flooding stress and adaptation mechanisms, as well as the stage after the advantage the breeding of rice varieties resistance to stress, laid a solid technical foundation... |
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