Cloning And Functional Characterization Of MiR2118b-5p Gene From Wheat (Triticum Aestivum L.)

Gametocidal Chromosome(GC)can induce chromosomal breakage,thereby causing chromosome recombination.The chromosome plays an important role in Wheat genetics and breeding.However,the mechanism of GC is still unclear so far.The CS-3C gametocidal chromosome monosomic addtion line(CS-3C)were created in our previous study,The anther of the CS and CS-3C were sequenced through high-throughput miRNA sequencing aimed to identify the miRNA related to the gametocidal.We found that miR2118b-5p differentially expressed.Speculated that miR2118b-5p may play a role in GC and elucidate the function of miR2118b-5p in wheat fertility.In this study,the pre-miR2118b-5p(MIR2118b-5p)was cloned and its function was studied.The main research results are as follows:(1)According to the structural characteristics of the microRNA precursor gene,primers were designed successfully from wheat genome.(2)The plant expression vector pOx-MIR2118b-5p was successfully constructed,which contained pUbi promoter and hygromycin resistance.(3)MIR2118b-5p genes was transferred into the rice plant by Agrobacterium tumefaciens,and 13 plants of hygromycin(Hyg)resistant rice were obtained.(4)Resistant plants were molecular biology detection by PCR and RT-PCR,6 transgenic rice plants were obtained,use of stem-loop qRT-PCR to detect miR2118b-5p mature expression in transgenic plants.The results showed that the 3 transgenic plants miR2118b-5p expression was significantly increased.(5)The predicted target gene OsSAUR36 was analyzed by qRT-PCR.The results showed that OsSAUR36 expression in transgenic rice was significantly lower than that in wild type.OsSAUR36 and MIR2118b-5p were negatively regulated in transgenic rice plants.(6)The pollen germination of the transgenic plants was lower than that of the wild type plants,and the seed setting rate of the transgenic plants decreased(7)compared with the wild type plants,and many others morphological changes were caused.