Functional Analysis Of Soybean MiR172 And Target Gene Involved In Floral Induction And Stress Response

Soybean is originated in China with a history of over 5000 years,and had been widely cultivated throughout the world at present.Soybean is an important food crop in our country as an important source of plant oil and protein.It occupied an important position in agricultural production throughout the world.Soybean is very sensitive to light and other natural condition,which characteristic affects the adaptation and yield of soybean varieties.As an important limiting factor that affect plant growth and development,abiotic stress such as drought,saline and alkaline stress could threaten crops in each period of growth,resulting in significant reduction in the yield of crops.In order to expand the soybean-growing areas in our country,improving the adaptability of soybean varieties,and further increasing the yield of soybean,we should change the flowering habit of soybean to make soybean flower and mature normaly in most regions.To strengthen the ability of soybean resistance to adversity stress,enhancing the stability of soybean production.micro RNA(miRNA)is widely distributed,endogenous and 20-24 nt noncoding single-stranded small molecule RNA in plants and animals.miRNA mediates the degradation of target genes.The transcriptional gene silencing causes the down-regulation of the expression of the target gene.In recent years studies have shown that plant miRNAs not only play an important role in the regulation of growth,development and morphogenesis process,but also be involved in biotic and abiotic stress response.The miR172 family is a conserved miRNA that has been found and studied in several plant species.Most research has shown that miR172 is involved in plant flowering time control.A family of AP2-like transcription factors acts as a repressor of flowering and has the common predicted target of miR172.Recent studies have shown that miR172 is also involved in stress responses.The miR172 family in wheat(Triticum aestivum L.)is increased in abundance under osmotic,salt and cold stresses.In rice(Oryza sativa L.),it was significantly decreased under drought stress.In the present study,we analyze the spatial and temporal-specific expression of gma-miR172 a and its target gene Glyma03g33470 in Dong Nong 42 by real-time RT-PCR.We analyze the role of gma-miR172 a and Glyma03g33470 in floral induction of soybean through the gma-miR172 a transgenic plants and toe1 complemented plants.In this study,real-time RT-PCR was also used to analyze the expression pattern of soybean gma-miR172 c and its target gene Glyma01g39520 treated with various stresses and exogenous hormones in Dong Nong 50.gma-miR172 c was over-expression in the model plant Arabidopsis thaliana.The phenotype and molecular mechanism of transgenic lines were analyzed to identify the function of gma-miR172 c in plant abiotic stress response.The main results were listed bellow:1.Real-time RT-PCR analysis showed that the abundance of gma-miR172 a increased throughout the entire vegetative phase,peaked at the late vegetative phase and formation stage of the flower bud.The expression pattern of gma-miR172 a has the specificity of time and space.2.5'RACE assays indicated gma-miR172 a directs Glyma03g33470 m RNA cleavage in vivo.Real-time RT-PCR analysis showed that the expression pattern of Glyma03g33470 has the specificity of time and space.3.The p CAMBIA3301-gma-miR172 a vector was used to transform into Arabidopsis,the transgenic plants were screened and identified by PPT resistance,PCR and real time RT-PCR,resulting in homozygous lines of T3 transgenic Arabidopsis with over-expression of gma-miR172 a.The p CAMBIA3301-r Glyma03g33470 vector was used to transform into Arabidopsis toe1 mutant,the complemented plants were screened and identified by PPT resistance,PCR and real time RT-PCR,resulting in complemented lines of T3 toe1 mutant with over-expression of r Glyma03g33470.4.gma-miR172 a transgenic plants flowered earlier than wild-type(WT)plants and had less leaves.The flowering time of toe1/35S::r Glyma03g33470 was between WT and toe1.Real-time RT-PCR analysis showed that gma-miR172 a up-regulated the expression of FT?AP1 and LFY to promote flowering.5.Real-time RT-PCR analysis showed that the expression level of mature gma-miR172 a,Gm DCL1 and Gm SE in Gm GIa-overexpression soybean were higher than Dong Nong 50.It demonstrated that GI can regulate miR172 maturation(processing)in soybean.6.Real-time RT-PCR analysis showed that gma-miR172 c could be induced by ABA,drought and salt.7.5'RACE assays indicated gma-miR172 c directs Glyma01g39520 mRNA cleavage in vivo.Real-time RT-PCR analysis showed that Glyma01g39520 could also be affected by ABA,drought and salt.8.The p CAMBIA3301-gma-miR172 c vector was used to transform into Arabidopsis,the transgenic plants were screened and identified by PPT resistance,PCR and real time RT-PCR,resulting in homozygous lines of T3 transgenic Arabidopsis with over-expression of gma-miR172 c.The p CAMBIA3301-r Glyma01g39520 vector was used to transforme into Arabidopsis snz mutant,the transgenic plants were screened and identified by PPT resistance,PCR and Real time RT-PCR,resulting in lines of T3 snz mutant with over-expression of r Glyma01g39520.9.The overexpression of gma-miR172 c in Arabidopsis remarkably increased the germination rate,cotyledon greening and root length compared with WT under treatment of mannitol and Na Cl.Detached leaves of gma-miR172 c transgenic lines lost water more slowly than those of WT.In addition,gma-miR172 c transgenic lines were hypersensitivity to ABA.Stress-responsive genes,proline content and activity of SOD in transgenic plants were up-regulated.These results suggest that gma-miR172 c is a positive regulator of salt and water deficit tolerance.10.Phenotype analysis showed that gma-miR172 c transgenic lines flowering earlier than WT especially under drought.11.The phenotype of snz /35S::r Glyma01g39520 under drought and ABA stress were between snz mutant and WT.It indicated that Glyma01g39520 can restore the phenotype of snz under ABA and drought conditions,proving that Glyma01g39520 is a functional target of gma-miR172 c in the stress response.