The Molecular Mechanism Of Regulating Seed Vigor By MiR164c And MiR168a

Seed is considered to be the optimal material for plant germplasm conservation. However, in the process of storage, seed vigor will gradually lose. It is absolutely important to explore a method to maintain seed vigor during its storage process. miRNAs are a class of endogenous, noncoding small RNA with 21-24 nucleotides that have recently emerged as a key regulator of gene activity, mainly promoting the cleavage the complementary target mRNA sequence or inhibiting its translation. In plants, through regulating the expression of target genes, miRNAs play an important role in all aspects of plant growth and development, including the morphogenesis of root, leaf and flower, the cell differentiation, the formation of transfusion tissue, and so on. They also participate in the feedback regulation of the synthesis of other small RNAs.In this study, seeds of conventional rice Kasalath and its of miR164 c, miR168 a overexpression or silencingtransgenes were taken as material, we explored the relationship between the expression of miR164 c or miR168 a and the vigor of seeds which were preserved in natural open storage condition or with artificial aging treatment. By means of bioinformatics, some of the target genes and their function of miR168 a and miR164 c were analyzed, and verified by RT-qPCR. Based on the above analysis, we proposed a hypothetical molecular mechanism of mi R164 c and mi R168 a regulating seed vigor via target genes. The main results are as follows:1. By testing the change of germination percentage of seeds of the conventional rice Kasalath and its miR164 c, miR168 a overexpression or silencingtransgenes preserved in natural open storage condition for 0 months, 8 months and 14 months, we found that in the natural aging process, the descent rate of germination percentage of mi R164 c silencing transgenes and miR168 a overexpression transgenes was postponed to the wild-type seeds, it indicates the anti-aging ability of seeds of miR164 c silencing or miR168 a overexpression transgenes is stronger than the wild-type seeds; while the descent rate of seeds of miR164 c overexpression transgenes and miR168 a silencing transgenes was faster than the wild-type seeds, it indicates that the anti-aging ability of seeds of miR164 c overexpression transgenes and miR168 a silencing transgenes is weaker than the wild-type seeds; Along with the storage, this difference was more significant, Under artificial aging conditions, the similar results were obtained. Therefore, it was suggested that upregulation of miR164 c expression leads to reduce seed anti-aging ability, while the upregulation of miR168 a expression is conducive to maintain seed vigor.2. Under artificial aging condition, the vigor of rice Kasalath and its all transgenic seeds declined gradually, characterized with the increasing relative conductivity of the immersion of seeds in water. It is worth noting that, after 8 days of aging, the conductivity of miR164 c silence transgenic seeds or miR168 a overexpression transgenic seeds was lower than the wild-type Kasalath seeds, while the conductivity of miR164 c overexpression transgenic seeds or miR168 a silence transgenic seeds was higher than the wild-type Kasalath seeds. This is consistent with the results of the germination testing.3. RTqPCR analysis further validated that miR164 c expression was negatively correlated with seed vigor, miR168 a expression was positively correlated with seed vigor.4. Based on bioinformatic analysis, PM27 is one of the potential target genes of miR164 c, AGO1 and PTR2 are two potential target genes of miR168 a. RTqPCR assay validated that in the seeds with lower viability, the expression of PM27 decreased, in accordance with the expression level of miR164 c. And miR168 a also presented a certain ebb and flow relationship with AGO1 and PTR2 expression level. Configuration analysis of PM27 and PTR2 showed that all of them may be some membrane proteins. It suggests that miR164 c and miR168 a may regulate these target genes, leading to changes in the structure and nature of biofilm systems, thus affects the vitality of seeds.