Cloning Of A MIR414 Gene And Preliminary Study On Its Stress-Resistance Function In Vaccinium Spp.

Blueberry,scientific name Vaccinium spp.,is an economically important berry fruit crop.Due to the richness of health-beneficial bioactive compounds such as anthocyanins,the blueberry-related researches have attracted more and more attention from all over the world.Blueberry has extreme limitation of root distribution with the lack of root hairs,leading to weak absorbing capacity.The abiotic stresses such as drought and salinity seemingly become major unfavorable conditions during blueberry fruit productivity.Thus,it is of significance to dig out stress-resistance regulators and conduct breed improvement accordingly.mi RNAs are a class of approximately 21 nt in length,non-coding RNA,and increasing evidences indicate that mi RNAs play important roles in response to various stresses.mi R414 is conserved across plant species,and it has been predicted to be involved in plant response to stress.However,the functions of mi R414 have not been experimentally revealed in plants.Previously a MIR414 gene was predicted using EST-based computational identification in our lab.In the study,the MIR414 gene from Vaccinium ssp.“Northland” was cloned and then transferred into the model plant Arabidopsis.Thus,the transgenic Arabidopsis was used for the study on the functional roles of MIR414 gene in response to stresses,and the detailed results are as follows:1.The genomic DNA was extracted from blueberry leave,and the primers with the restriction sites of Bam HI and Sac I were designed in term of the corresponding EST sequence.MIR414 gene was successfully cloned,and the PCR products showed similar size to the expected ones with 361 bp.2.The target gene fragments were constructed into the binary vector pBI121,and the p BI121-MIR414 was obtained accordingly.Subsequently,the gene was introduced into Arabidopsis using Agrobacterium-mediated transformation approach.3.Positive transgenic lines were screened using kanamycin,followed by PCR confirmation.The positive transgenic lines were transferred and grown for collecting seeds of T1 generation.The hemizygous seeds of T3 generation were used for the following study.4.The T3 hemizygous seeds of transgenic lines and WT were planted onto MS medium with 0 mmol / L,50 mmol / L,100 mmol / L,150 mmol / L Na Cl,and the seed germination rate and green rate of seedling were investigated for transgenic lines and WT,respectively.It was observed that transgenic lines showed higher germination rate as compared to WT under salt stress,and the difference of germination rate between transgenic lines and WT was enhanced as the concentration of Na Cl increases.When exposed to150 mmol / L Na Cl,the germination rates of two transgenic lines were up to 81.63% and 90.67%,respectively,while only 57.74% germination was observed for WT.However,green rate of transgenic seedlings was lower than the ones of WT under salt stress,and the difference of green rates of seedlings between transgenic lines and WT was enhanced as the concentration of Na Cl increases.When exposed to150 mmol / L Na Cl,only 13.77% and 20.13% green seedlings were observed for two transgenic lines,respectively,while the green rates of WT was up to 47.05%.5.The T3 hemizygous seeds of transgenic lines and WT were sowed onto MS medium with 0 mmol / L,100 mmol / L,200 mmol / L,300 mmol / L Mannitol.It was observed that the germination rates of transgenic lines were higher than the ones of WT in the presence of 300 mmol / L Mannitol.The germination rates of two transgenic lines reached to 95.64% and 97.16%,respectively,while only 83.64% germination was observed for WT.However,green rate of transgenic seedlings was lower as compared to the ones of WT in the presence of 300 mmol / L Mannitol.It was observed that two transgenic lines showed 57.38% and 48.56% green seedlings,respectively,while the green rates for WT was up to 72.09%.Collectively,our findings indicated that MIR414 can confer the seed germination and depress seedling growth to some degree under salt and mannitol-stimulated drought stress,which will lay a foundation for further studying the functional roles of MIR414 in response to abiotic stresses,and also provide a reference for studying the functional roles of MIR414 in other plant species.