Construction Plant Binary Vector Of STTM-OsmiRNA And Transformation Of Rice Callus

Rice as the main food in the world,improve the yield and quality of rice,not only has the vital significance to national food security,also is good for people's health.Rice has an extensive adaptability in our country,and the planting area is about from the northeast to the southwest,suggesting that the rice has extensive adaptability to the environment.Rice grain yield is complex quantitative traits,depends on it yield characters,and plant growth and development also has influenced on the grain yield.Although,we have already separated and cloned some gene about rice grains developmen,and it is important for us to explore the function.However,the research of micro RNAs(miRNA)in rice growth and yield formation is poor.Mi RNAs are small non-coding RNA,and play an vital role in plant growth and development and yield formation.Osa-miR319 family contains two members,and their related studies shown that the fuction of miRNA were not only involved in plant growth and development,but also associated with biological and abiotic stress responses in plants.In this study,we employed a powerfull technology,named short tandem target mimic(STTM)to knock out OsmiR319 family members by agrobacterium-mediated genetic transformation of rice callus,and then screening positive transgenic mutants,and analysis the function of OsmiR319 in seeds developing.In brief,the main results were summarized as below:(1)In our study,we employed PCR amplification to constructed six plant binary vector and they are STTM319,STTM390,STTM397,STTM398,STTM1432 and STTM528.First,according to the STTM stem-loop structure design of a pair of back-to-back primers,and then Take the plasmid p OT2-poly-cis as the template for PCR amplification,and the amplification products is about 3280 bp,after then,purified PCR amplification products,then digest by Swa I,ligation,transformation and identificated the recombination plasmid p OT2-STTM319 by sequencing.The subcloning vector p OT2-STTM319 needed to adding Pac I loci by PCR amplification,and the PCR products is about 2800 bp,after purified and co-digested with p CAMBIA1300/Pac I by Pac I,ligation,transformation,sequencing the binary vector,then we got STTM319 plant binary vector,and lt could be used to transformated into agrobacterium for transformation of rice callus.(2)We used histochemical staining,PCR amplification and fluorescence quantitative PCR to identification the regenerated plants.The root of positive seedlings in GUS buffer incubate for 0.5-3h will become blue,witch indicated that they are maybe positive seedlings,after detective the relative expression of miR319's primary transcription and it mature miRNA,we find that it is decreased 3~5 times compared with the wild type.It is interesting that the height and the tiller number is decreased,and they are 84.9cm,and 7.5 respectively.Also,about 6.6mm,3.1mm and 16.9g in the grain length,grain width and grain weight,difference reached significant level.(3)Due to the phenotypes of STTM319 reduced in grain size,grain weight and plant height,therefore,we will focus on the aspect of grain size and grain weight to carry out follow-up study.Then we will employed small RNA sequencing and transcriptome sequencing to explore regulation network and the main genes,to find out molecular mechanism of miR319 in grain filling.