Screening And Identification Of MiRNAs Involved In Transgression Of Differeniating Capacity In Rice Hybrids Calli

Rice(Oryza sativa)is one of the most important economic crops for annual grasses and one of the most successful crops used in agricultural production.Hybrid rice significantly increased the per unit area yield,and solved the food crisis to some extent.However,the offspring of hybrids would have trait separation and cannot maintain heterosis.The annual breeding work consumes manpower,material resources and financial resources.While,the mechanism of heterosis has not been clear so far.In this experiment,we compared the miRNAs differences of callus between pure line parents Nipponbare,93-11 and its reciprocal crosses,and searched for related miRNAs which involved in the regulation of the callus differentiation capacity of the F1 generation.We also analyze the tissue anatomy of parental and hybrid callus,and explain the miRNAs in rice from the histological and molecular level.In order to explain the regulation mechanism of transgression of difFereniating capacity in hybrid callus,we performed a series of research as follows.(1)Histological observation of rice callus.Using rice mature embryos of pure line parental Nippon bare(japonica),93-11(indica)and its reciprocal cross hybrid N9(Nip is the female parent,93-11 is the male parent),9N(93-11 is the female parent,Nip is the male parent)as explants to induce callus and differentiate them.We counted the induction rate and differentiation efficiency.At the same time,the paraffin section technique was used to observe the cell characteristics of different genotype callus.The results showed that the callus cells of rice hybrids were arranged in a large number of parenchyma cells,and a large number of cell clusters with strong meristems were distributed on the outside,which laid a foundation for the efficient differentiation of hybrids.The cells of parental callus,especially the 93-11 callus,have irregular cell distribution and are severely deficient in cell clusters with meristematic ability.Most of the cell walls are lignified or corked,which may restrict the differentiation of callus.(2)Screening and identification of candidate miRNAs in callus differentiation ability of rice hybrids.Illumina solexa high.throughput sequencing technology was used to screen differentially expressed miRNAs in hybrids and stimulation hybrids.And we analyzed the function of potential target genes.A small number of candidate miRNAs were screened and analyzed as a target for subsequent validation analysis.The results confirmed that under tissue culture conditions,the expression of miRNAs between hybrids and pure line parents showed significant differences,involving a large number of metabolic pathways for cell regeneration and reproduction.(3)Functional verification and analysis of candidate miRNAs.The silencing vector of candidate miRNAs was constructed by STTM technology,and the transgenic plants of rice were obtained by Agrobacterium transformation.The callus analysis induced by mature embryos of transgenic plants showed that the transgenic callus differentiation ability decreased significantly.The callus anatomy analysis showed that the silencing of Osa-miR396 resulted in changes in the microstructure and morphology of rice callus.It was further confirmed that Osa-miR396 is involved in the transgression of differeniating capacity in rice hybrids calli.At the same time,the transgenic plant STTM-Osa-miR396 was crossed as the female parent with the male parent 93-11,and the hybrid STTM-Osa-miR396/9311 was obtained.The expression of miR396 and its target gene was analyzed by qRT-PCR.It was observed that the differentiation ability of STTM-Osa-miR396/9311 was greatly reduced compared with the hybrid N9.