| Corn is one of the most important crops in the world, in the total grain output around the world, the total corn production ranks first, and trade volume ranks second. To increase the yield of maize is years of scientific breeding workers together to target. "Late spring coldness" is one of the main disasters of maize production in Northeast Chinese. The sowing date suffered low temperature, which would delay the emergence of maize, and seriously affect the yield of maize. The Arabidopsis thaliana seedlings were used as material to extract the total RNA, and the ICE1 gene was amplified by RT-PCR to complete the development of the reading frame sequence. The recombinant plant expression vector pCAMBIA3301- ICE1 was constructed. The gene was transferred into the tobacco through agrobacterium mediated transformation. Positive transgenic plants were obtained through PCR detection of transgenic plants. Through the detection of Southern blotting detection, physiological and biochemical indexes and real-time fluorescence quantitative PCR detection of the transgenic, positive plants cold resistance of Arabidopsis thaliana ICE1 gene was stronger than that of the untransformed plants; By using Agrobacterium mediated method into maize inbred lines in H99 plants, cold tolerance positive plants were obtained, to lay a foundation for the cultivation of New Maize Germplasm with cold tolerance.The main research results obtained are as follows:1. The complete open reading frame of ICE1 gene sequence was cloned from wild type Arabidopsis thaliana, and constructed the recombinant pCAMBIA3301- ICE1-Bar vector for plant expression through the seamless cloning technology.2. Tobacco leaf discs were transformed by Agrobacterium mediated method. 9 positive plants were obtained by PCR detection, and the conversion rate is 15.5%. Southern blotting test results proved that the ICE1 gene was integrated into tobacco genome, as the transgenic tobacco were treated in low temperature environment(4°i). Its relative conductivity reduced 12.00%~17.65%. Proline content increased 13.28%~24.10%. MDA content reduced 7.09%~14.52%, peroxidase activity 9.39%~22.54%. The mRNA expression of the ICE1 gene were found in the organ root, stem and leaves of the transgenic maize detected by using the Real time fluorescent quantitative PCR assay(qPCR), and particularly in the leaves.3. Using orthogonal experimental design L9(34), the immature embryos of 3 maize lines were used to study the genotype, young embryo size, concentration of 2,4-D and concentration of AgNO3 on the callus induction. Aanalysis results show that the effect size of various factors on callus induction followed by genotype>immature embryo size>2,4-D concentration>AgNO3 concentration. The optimal combination was H99, and immature embryo size was 1.5-2mm. 2,4-D concentration was 2mg/L and AgNO3 concentration was 8mg/L.T he callus induction rate was 81.35%.4. Through the method of Agrobacterium mediated transformation of maize inbred line H99 embryogenic callus, positive transgenic plants were obtained and transformed. 3 T0 transgenic plants were obtained through agrobacterium mediated method, and the transformation rate is 1.06%. 21 seeds of T0-generation were harvested. After the indoor planting, 7 T1 positive plants were obtained.5. Test results of H99 T1 Southern blotting generation plant proved that the ICE1 gene was integrated into maize genome, as the transgenic maize were treated in low temperature environment(4°i). Its relative conductivity reduced 23.71%~33.73%. Proline content increased 10.68%~20.16%. MDA content reduced 13.60%~18.60%, and peroxidase activity increased 17.40%~27.45%, compared with the control. The expression of ICE1 gene increased the cold tolerance of plants. The mRNA expression of the ICE1 gene were found in the organ root, stem and leaves of the transgenic maize detected by using the Real time fluorescent quantitative PCR assay(qPCR), particularly in the leaves. |
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