| Protein Disulfide Isomerase (PDI) are multifunctional enzyme proteins which widely exists in eukaryotic organisms. They are involved in the catalysis of disulfide oxidation reduction reaction, rearrangement mismatch disulfide bonds, and in the nascent peptide synthesis, processing and transportation into a mature polypeptide of correctly folded. Not only they have close contact with the nitrogen metabolism of plant grain organs, but play an important role in the stress on physiological processes. In this paper, Nipponbare was used as the receptor material to construct PDI gene silencing and overexpression vector respectively and the transgenic plants were obtained through the transformation of the callus. The PDI gene silencing and overexpression plants were determined on plant phenotype, fruiting characters and some important quality indexes; and based on the temperature controlled processing test, the transgenic plants were studied on the expression of some related gene and the relationship between heat resistance physiological indicators of rice. The main results are as follows:1. The bioinformatic analysis showed that PDI gene was highly conservative in rice, especially its catalytic function domains called a and a’.Then the vectors of pTCK303-RiOsPDI and pTCK303-OsPDI were successfully constructed and was transformed into the wild type Nipponbare by agrobacteria-mediated method. After selected by the PCR technology of amplifying the Hyg gene fragment, we got the transgenic rice plants. RT-PCR and RT-qPCR results showed that the expression of PDI gene was significantly reduced in PDI gene silence plants in different organs, especially in seeds; And the expression has been at a very low level in different filling stage through q-PCR detection. Whereas the PDI overexpression plants showed the opposite trait that the expression of target gene was higher than controlled Nipponbare relatively. The results of analysis in physiological and ecological characteristics in rice seeds indicated that PDI gene silencing can declined the rice grain weight and increased the chalky grain rate and chalky degree, but it is no dfference in total protein content, amylase and protein components(albumin, globulin, gliadin and glutenin). PDI gene silencing decreased the total protein content, increased the amylase content and change the contrent of four protein components.2. In addition, through the high temperature treatment in growth chambers, it would be taken the samples at different times in order to detect the expression of related heat shock protein gene in the filling stage. The results said that PDI gene would be induced to upregulate and increased the chalky grain rate and chalky degree under high temperature stress, the test material(PDI silence, overexpression and wild-control Nipponbare) showed a similar trend, but PDI silence would reduce the heat resistance of rice, declined the seed rate greater than overexpression or wild-control Nipponbare and relatively increased the chalky grain rate and chalky degree PDI silence could increase the gene expression of heat shock proteins such as Bip, Hsp70and Hsp90etc.especially a greater impact on Bip gene showing "complementary effect" obviously, while PDI overexpression to some extent inhibited the expression of Bip gene, but not significant as PDI silence on heat shock protein; furthermore, PDI silence and overexpression also had effects on soluble pretein, MDA and proline accumulation under high temperature stress during grain filling stage, but the relationship is more complex. |
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