| Soybean(Glycine max L. Merr.), originated from China with a long cultural history, is one of main source of protein and oil. Because of the serious influence to soybean production induced by drought and salt stress, it is necessary to research the function of stress-response genes, which would has important theoretical and application value to reveal the mechanism of soybean drought-resistant and cultivate drought-resistant varieties. In this study, we aimed to understand the function of soybean genes and mi RNA members using the CRISPR/Cas9 site-directed mutagenesis system constructed by us. And we analyzed the biological function of soybean gma-mi R160, which was expected to offering important information for drought stress tolerance. The main results were listed as followed.1 Construction of CRISPR/Cas9 site-directed mutagenesis system in soybean. In this study, ten U6 genes were firstly predicted in soybean through bioinformatics, and two vectors(p Cas9-Gm U6-sg RNA and p Cas9-At U6-sg RNA) were constructed using the soybean U6-10 and Arabidopsis U6-26 promoter, respectively, to produce sg RNAs. Three genes, glyma06g14180, glyma08g02290 and glyma12g37050, were selected as the targeted mutation genes. We compared the two types of vectors through both the transformation into soybean protoplasts and soybean hairy roots by Agrobacterium rhizogenes infection. and found that base substitutions were usually appeared in soybean protoplast transformation, gene deletions and insertions were inclined to be produced in soybean hairy roots. As for the transformation into soybean hairy roots, mutation efficiency induced by the p Cas9-Gm U6-sg RNA vector had a higher mutation rate(14.7-20.2 %) than that of the p Cas9-At U6-sg RNA vector(3.2-9.7%). Besides, off-target associated with glyma06g14180 and glyma12g37050 were also detected both in the transformation of soybean protoplasts and soybean hairy roots. All the results indicated that targeted mutagenesis by the CRISPR/Cas9 system could be a preferred method to research the function of soybean gene, especially these genes involved in roots and nodules.2 The application of CRISPR/Cas9 system to obtain the mutation of ath-mi R160 members. To understand the function of The specific sg RNA guide sequences were designed based on the precursor sequences of the three members ath-mi R160a/b/c. The sg RNAs were obtained with T7 RNA synthesis kit. Then we proved that the fragments of DNA containing the precursor sequences of ath-mi R160a/b/c could be cleaved by Cas9 nuclease in vitro. What is more, single-base mutant in the precursor of ath-mi R160 c were found in Arabidopsis protoplast. All the results suggested that several mutation in the precursors of ath-mi R160 family could be obtained through the transformation of Arabidopsis. which may lay foundation for researching the functions of mi R160 and other mi RNA members.3 Research about the drought stress response of soybean gma-mi R160. Using a 5’ RNA-ligase-mediated Rapid Amplification of c DNA Ends assay(5’RLM-RACE), we identified that eleven of twelve predicted target genes of gma-mi R160 were cleaved, and the cleavage sites were located at approximately the tenth or eleventh nucleotide of the target genes sequences relative to the 5’-end of mature gma-mi R160. The results of transient expression showed that the relative expression of glyma04g43350.1, glyma10g35480.1 were regulated by gma-mi R160. The results of q RT-PCR indicated that gma-mi R160 and one of its target glyma10g35480 was expressed differently in soybean tissues, various developmental stages and induced by drought. Overexpression of gma-MIR160 a showed that the roots of transgenic plants were shorter than those of WT plants on the MS medium containing 100 m M mannitol. And the transgenic plants began to wilt firstly and were turned to be normal later than WT, which suggested that gma-mi R160 might be a negative regulator of drought tolerance in soybean. The results provided important value about the mechanism of mi RNA responding to drought stress. |
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