Study On The Gene Silencing Of Plant-type PEPCs By Artifical MicroRNAs In Arabidopsis Thaliana

As one of cytoplasmic enzymes, Phosphoenolpyruvate carboxylase(PEPCase) carries out a variety of physiological functions in vivo. It is widely distributed in higher plants, algae and most bacteria. In C4 and CAM plants, phosphoenolpyruvate carboxylase plays an important role in the initial CO2 fixation in photosynthesis. In C3 plants, PEPCase is proposed to be a key enzyme to control the ratio of protein/lipid due to its key contribution in compensating intermediate metabolites in tricarboxylic acid cycle, and balancing carbon/nitrogen metabolism. Meanwhile, PEPC may be closely related to the environmental stress on the plants.Arabidopsis thaliana and oilseed rape (Brassica napus L.) belong to Cruciferae, Brassica. They have close genetic relationship, existing on average about 85% similarity in the coding region of the genomes. Therefore, studies on A. thaliana provide important reference for rapeseed breeding.To investigate the effects of plant-type PEPCs on modifying lipid content, fatty acid composition and the response to environmental stress in A. thaliana, the plant-type PEPCs genes of A. thaliana were designed to be silenced by the artificial miRNA (amiRNA).We constructed the plant expression vector and transformed it into wild type A. thaliana (col-0) by the floral dip method. In this way, we successfully accquired transgenic plants. The related work of molecular identification and genetic analysis was carried out.Main results of this study are followed:1. We input the cDNA sequence of Atppc1, Atppc2 and Atppc3 into the website of WMD3 to design the artifical miRNAs online. Then, we synthesized the related primers to clone the precursor fragment of amiRNA by overlapping PCR, with the pRS300 plasmid as incipient template, which contains the precursor backbone of miR319a, one of the endogenous miRNAs in A. thaliana. Subsequently, we cloned the precursor fragment of amiRNA into the binary expression vector of pFGC5941 to obtain the plant transformation vector, named pFGC-amiAtppc123, and then transformed it into A. thaliana by the floral dip method.2. With the herbicide basta spraying, most To generation seedlings withered, but still some of them grew normally. We chose 18 normally growing plants for molecular assessment. All of them were successful transformants, which were confirmed by PCR, with the leaves genomic DNA as templates. The amiRNA transcription levels were assessed by semi-quantitative RT-PCR. The results showed that amiRNA transcription level was drastically increased in transgenic plants compared with non-transgenic plants. The results of semi-quantitative RT-PCR showed that the intact mRNA level of Atppcl was drastically suppressed in transgenic plants, compared with non-transgenic plants. However, the expression of Atppc2 and Atppc3 were not significantly changed.3. Under the stress of NaCl, Atppcl and Atppc3 showed different transcription profile between negative control and transgenic plants. Nevertheless, transcription of Atppc2 was relatively stable. Assessment on the T2 generation transgenic seeds showed oil content of transgenic plants had no significant improvement, compared with non-transgenic plants, but fatty acids composition was slightly adjusted with higher low-carbon saturated fatty acids content.