Preliminary Study On The MiR156-mediated Mechanism That Controls Seed Maturation In Arabidopsis Thaliana

The stage of accumulation of seed storage at seed maturity is a critical period for determining seed quality and yield.mi RNAs have a central position in the gene regulatory network.However,the mi RNA-mediated regulation of seed maturation is still not well known.In the present study,a laboratory-built detection system(gus23,a GUS gene is driven by a seed-specific promoter)was used to screen the mi RNAs involved in the regulation of Arabidopsis seed maturation.Our study indicated that mi R156 and its targets(SPL2,SPL10,SPL11)might play important roles in the regulation of seed maturation.The main results are as follows:1.Through literature search,we found that some mi RNAs might be involved in the regulation of seed maturation.Based on the mi RNAome data of seed development,mi R156,mi R397,mi R402 and mi R824 were selected for further study.The four pre-mi RNAs were cloned,constructed and transferred into gus23.2.MIM156,a down-regulated mutant of mi R156,was crossed with gus23,and GUS-staining analysis indicated that the real leaves of 14 d seedlings showed GUS phenotype.Additionally,the size of seeds in MIM156 was bigger than the ones in wild type.These results suggested that mi R156 might be involved in the regulation of seed maturation in Arabidopsis.Meanwhile,mi R156-overexpressed mutant and down-regulate muatant also showed other phenotypes.For example,over-expression of mi R156 caused late flowering,more rosette leaves,dwaf,short silique,less seeds.3.It has been demonstrated that mi R156 family is comprised of 10 members(mi R156a-j).To provide information for dissecting functional roles of differant mi R156 members,the expression patterns of 10 mi R156 s were investigated in seedling,rosette leaves,flower and silique.As a result,the majority of mi R156 familyshowed higher expression in silique and flower.These results indicated that mi R156 s might perform important functions during development of seed and flower in Arabidopsis.4.It has been well-known that mi R156 can negatively regulate the expression of SPL family.Gene expression data in different tissues retrieved from TAIR indicated that SPL2,SPL10 and SPL11 showed higher transcriptional accumulation in seeds.Thus,we cloned SPL2 and transferred into gus23,while over-expression mutants for SPL10 and SPL11 bought from ABRC were crossed with gus23.GUS-staining assay indicated that the over-expression mutants of SPL10 and SPL11 showed GUS phenotype on the true leaves of seedling,while GUS phenotype was observed on the cotyledon of over-expression mutant of SPL2.Furthermore,the transcriptional accumulations of four key regulators of seed maturation(LEC1 ?LEC2?ABI3 and FUS3)were examined in the seeds of over-expression mutant of SPL10 and SPL11,significant enhancements were observed for ABI3 and FUS3 as compared to wild type.These results suggested that SPL2,SPL10 and SPL11 might involved in the regulation of seed maturation as the targets of mi R156.5.To address the functional roles of mi R156 in the regulation of seed maturation,RNAi approach was used to silence SPL10 and SPL11.Subsequently,the phenotypes of over-expression mutants and down-regulated mutants were further observed.These findings in the study first explored the functional roles of mi R156 and its targets in the process of seed maturation,which laid a foundation for addressing mi R156-mediated mechanism that controls seed maturation,and provided a reference for developing mi R156-related research in other plant species.