| Rice was one of the most important food crops in China, it made an important contribution to ensuring national food security. In recent years, “a hundred billion kilograms of grain production project” was raised by Heilongjiang Province, The high yield and stable production of rice was an important guarantee. However, Heilongjiang province is located in cold area, rice production is effected by chilling injury. Therefore, study on cold resistant gene and the function of its promoter in plant, it can not only provide the excellent gene and promoter with independent intellectual property rights for genetic engineering breeding of plant, but also lay the foundation for the research of anti cold mechanism of plant. The cold acclimation protein ThCAP gene of the Tamarix hispida(accession number: AY587773) in this test was a member of the COR413 gene family. The preliminary results show that overexpression of the gene can improve Populus(P.davidiana×P.bolleana)ability to resist cold stress, and the effect is remarkable, but its promoter in rice and its biological function is unknown. In this study, the function of this gene and its promoter were further studied by transgenic methods. The main results were as follows:1. The pCUbi1390-ThCAP plant expression vector was constructed. 26 transgenic ThCAP rice resistant plants were obtained by agrobacterium mediated method. 18 positive plants were detected by PCR. It proved that the foreign gene had been integrated into the rice genome and it can be normal transcription and translation into the correct protein by RT-PCR and Western Blot.2. The experiment of low temperature treatment on 5 T2 generation transgenic plants showed that: the proline content, MDA content and relative electrical conductivity of transgenic plants were not significantly different from that of non transgenic plants under normal growth conditions; the proline content of transgenic plants was significantly higher than that of non transgenic plants, MDA content, relative electrical conductivity of transgenic plants were significantly lower than those of non transgenic plants after 7d low temperature treatment. So,the expression of ThCAP gene increased the ability of transgenic rice to resist low temperature.3. The 1538 bp promoter candidate fragment of upstream sequence of ThCAP gene was cloned from Tamarix genome by genome walking. The sequence had a wide variety of plantuniversal promoter elements and a variety of cis acting elements associated with abiotic stresses by bioinformatics software prediction, it showed that the sequence had the general biological function of the promoter.4. According to the position of the cis acting element, 5’ terminal deletion fragment of GUS transient expression vector of ThCAP gene promoter was constructed, and it was transformed into Arabidopsis thaliana, GUS induced expression was performed at low temperature. The results of GUS histochemical staining showed that the 1538bp-900 bp promoter of ThCAP gene was a key regulatory region for the cold response. There were DREB/CBF, bZIP and MYB of cis acting element of transcription factor in this region. The results of this study could lay a foundation for further elucidation of the regulation pathway of ThCAP gene expression.This study through the ThCAP gene of rice genetic transformation and functional analysis,it is clear that the ThCAP gene over expression can improve the ability of transgenic rice resistance to low temperature. On the basis of, this paper and cloning the gene ThCAP promoter region, identified the promoter with general biological function of promoter, and through the series of deletion analysis of the promoter, suggesting that gene ThCAP start 1538 bp-900 BP response to cold key regulatory region. The cis acting elements of DREB/CBF, bZIP and MYB transcription factors are present in the region. The results of this study could lay a foundation for further elucidation of the regulatory pathway of ThCAP gene expression. |
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