Expression Analysis Of MiR398 And Identification Of Target Gene And Regulation Research From Soybean Under Stress

Micro RNAs are non-coding small-molecule RNAs which are widespread in eukaryotes and have regulation functions. Micro RNA starts with a Pri-mi RNA of stem-ring structure which will then be processed by the Endonuclease Dicer-like, and finally a mature mi RNA of about 19~24 Nucleotide long is generated. Through the difference of its complementary with m RNA of the target gene, the RNA induced silencing complex(RISC) to crack or inhibits the translation and expression of its target gene. In plants the mi RNAs have been involved in almost all biological metabolism processes. For example it plays a significant role in the biosynthesis of cell walls and the botanical response to stress. In this study the expression amount of mi R398 gene series in different tissues of soybean upon salt, alkali, salt-alkali, abscisic acid and drought treatments were via real-time qualitative fluorescence PCR method analyzed. With the help of online analysis software “ps RNATatget” the target gene was predicted, which was verified by the improved RLM-5’ RACE method. In the meanwhile the expression amount was analyzed by RT-q PCR method. And the sub-cellular localization of the target was carried out. On this basis, the Pre-mi R398 s gene was constructed onto plant expression vector p Basta-mi R398 s and Arabidopsis was converted. The gene functions of Gma-mi R398 a, Gma-mi R398 b and Gma-mi R398 c have been preliminarily analyzed.1. The expression levels of mi R398 gene series upon different stress treatments have been analyzed by real-time qualitative fluorescence PCR method. The results indicate that the expression amount of Gma-mi R398 c upon salt-alkali, drought, abscisic acid and salt stress have all undergone a course of dropping at 1h, increasing at 3h and dropping at 12 h in contrast with 0h. The expression amount of Gma-mi R398 c upon alkali treatment has notably increased at 1h and 3h and dropped 4.5 times at 12 h. The expression amount of Gma-mi R398 b has shown a tendency of increasing upon salt-alkali, drought, abscisic acid, alkali and salt treatment. Among them the expression amounts upon drought and salt-alkali treatment have raised by 4 times by 12 h in comparison of 0h.2. With the help of online analysis software “ps RNATatget” the target gene of Gma-mi R398 s was predicted and the cutting position of the target gene was verified by the improved RLM-5’ RACE method. The expression amount was analyzed by RT-q PCR method. The sub-cellular localization of its target gene was further carried out. The TC452415 gene was connected to p CAMBIA-1302 by Enzyme digestion method. The tobacco leave was instantaneously converted by agrobacterium injection. It was confirmed by observation through confocal microscopy that the target gene has specially expressed on the membrane.3. The Pre-mi R398 s gene of soybean was constructed onto the botanical expression vector p Basta with the promoter of 35 S and was employed in converting agro bacterium. And the Pre-mi R398 s gene of soybean has been applied in genetic transformation of Arabidopsis with Floral dip method and the T1 generation of Arabidopsis lines with Gma-mi R398 a, Gma-mi R398 b and Gma-mi R398 c are respectively 16, 15 and 24.4.The separation ratio sifting on T2 generation lines has been carried out and the single copy transgenic line with a separation ratio of 3:1 which is in approval of Mendel genetic law has been selected. And the single copy transgenic high expression lines of T2 generation were by real-time qualitative fluorescence PCR method sifted through.5. For further investigation on functions of mi R398 s gene of soybean, stress treatments on the selected high expression transgenic lines and wild Arabidopsis were carried out. The germination rate on mediums with salt and drought treatments indicate that the germination rate of transgenic Arabidopsis with Gma-mi R398 c is notably lower than that of control group and those with Gma-mi R398 a and Gma-mi R398 b. Upon drought treatment the germination rate of Arabidopsis with Gma-mi R398 c is notably lower than that of control group and those with Gma-mi R398 a and Gma-mi R398 b.