Prokaryotic Expression And Genetic Transformation Of SoNCED And SoSnRK2.1Gene Form Sugarcane

The arms of the present study were to clone the key gene SoNCED in ABA biosynthesis and the key gene SoSnRK2.1in ABA signal transduction, construct their prokaryotic expression vectors and detect their expressions in E. coli, purify and identify their expression products; and construct the SoSnRK2.1gene eukaryotic plant expression vector and transfer it into the Nicoliana tabacum by Agrobacterium-mediated method. The main research contents and results are as follows:1. Based on the previously cloned total length of the SoNCED and SoSnRK2.1genes, the specific primers were designed for restriction of the prokaryotic expression vector pET30a (+) and the plant expression vector PBI121. Using the leaf RNA of sugarcane under stress treatment, the ORFs of the genes SoNCED and SoSnRK2.1were cloned with PCR amplification. The sequencing analyses showed that the amplified ORF of SoNCED gene was about1827bp in size, that of SoSnRK2.1gene was about1002bp in size, which exactly matched to the full-length sequences.2. The prokaryotic expression vectors of the pET-SoNCED and the pET-SoSnRK2.1were constructed successfully, and the two genes could express their target proteins under37℃and1.0mmol/L IPTG induction. The prokaryotic expression vector pET-SoNCED recombined protein was about66kDa in size, and the prokaryotic expression vector pET-SoSnRK2.1recombined protein was about38kDa in size, which were consistent with the predicted protein size. The pET-SoNCED and pET-SoSnRK2.1recombined proteins existed as inclusion bodies, and a single protein strip was produced after purifying each of them with Ni2+-NTA column affinity chromatography, and the result of MALDI-TOF identification showed the isolated pET-SoNCED recombined protein was NCED protein. 3. Using real-time quantitative PCR technique to analyze the expression patterns of the genes of SoNCED and SoSnRK2.1under four stresses, the results showed that the expressions of the two genes could be induced by different stresses, but the expression amount was different in different processing time. It is resumed that the two genes play an important role in plant response to adverse stresses.4. The eukaryotic plant expression vectors of SoSnRK2.1gene were successfully constructed, and the directly inserted vector pBI121was named as pBI-2-SoSnRK2.1, and the reversely inserted vector pBI121was named as pBI-3-SoSnRK2.1. The recombined plant expression vectors were transferred into the Nicoliana tabacum by Agrobacterium-mediated transformation for verification.