Cloning And Functional Expression Of ACCase Gene In Vernicia Fordii

Vernicia fordii, as the unique non-wood tree in China, is listed as one of Chinese four oil plants with Sapium sebiferum, Juglans regia and Camellia oleifera. Because of great gloss, non electrical conductivity, imperviousness, anti-corrosion, heat resistance, its seed oil which is widely used in compositing coating and paint become an important industrial raw material. Fatty acid is an important component of oil, in the pathway of fatty acid synthesis, ACCase catalytics CoA into malonyl-CoA, and malonyl-CoA is not only reactant in the the pathway of fatty acid synthesis and fatty acyl strand extension system, but also involved in the synthesis of fatty acid as donor, so ACCase is considered as a basic Metabolic substrate in organism. Especially in fatty acid synthesis pathway, ACCase is the first step and key step, even limiting step. This thesis carrys out a number of studies about gene cloning of ACCase from Vernicia fordii, expression pattern in tissues. The major results are as follows.1.cDNA Molecule Cloning and Bioinformatics Analysis of ACCase from Vernicia fordii. ACCase is Be separated from tung tree saved in our laboratory, according to the transcriptome data of tung tree, we design four pairs of primers and use RT-PCR technology to clone four subunit genes’ full-length cDNA sequence. Biotin carboxylase full-length cDNA is 1605bp, coding 543 amino acids, the predicted protein molecular weight and isoelectric point is 58262.0 Da and 7.98, GenBank number is KF964573; biotin carboxyl carrier protein full-length cDNA is 813bp, coding 543 amino acids, the predicted protein molecular weight and isoelectric point is 26496.6 Da and 5.76, GenBank number is JQ736807; carboxyl transferase alpha subunit full-length cDNA is 2313bp, coding 770 amino acids, the predicted protein molecular weight and isoelectric point is 85902.7 Da and 8.48, GenBank number: KF964574; carboxyl transferase beta subunit full-length cDNA is 1506bp, coding 501 amino acids, the predicted protein molecular weight and isoelectric point is 56688.1 Da and 4.92.2.Genome Molecule Cloning of ACCase from Vernicia fordii. We extract dna from tung tree saved in our laboratory and design four pairs of primers. Then clone four subunit genes’ full-length genome sequence by using RT-PCR technology. AccC and accD does not contain introns. AccB’s genome sequence is 2157bp, containg 5 introns. location is 128bp-845bp,1001bp-1100bp,1419bp-1577bp,1648bp-1841bp, 1907bp-1991bp. AccA’s genome sequence is 4995bp, containg 9 introns. Location is 328bp-425bp,501bp-1465bp,1574bp-1728bp,1888bp-1998bp,2116bp-2199bp,2355bp-2017bp,3070bp-3148bp,3218bp-3577bp,3656bp-3821bp.3.The relative expression of ACCase Gene in different periods of seed development and different organs and tissues from Vernicia fordii. The analysis by using qPCR technology shows the relative expression of ACCase Gene in different periods of seed development and different organs and tissues from Vernicia fordii. In the development process of seed, four subunit genes both have 2 peaks of transcription(early september and mid-late october). AccC’s relative expression in petal and pistil; accB’s relative expression in petal, pistil and sepal; accA’s relative expression in petal, pistil and stamen; accD’s relative expression in young leaf are both larger than other organs and tissues.4. Eukaryotic expression of ACCase from Vernicia fordii. The plant expression vector pCAMBIA1304-35S-accA, pCAMBIA1304-35S-accB, pCAMBIA1304-35S-accC, pCAMBIA1304-35S-accD are constructed success-fully transfo-rmed into agrobacterium GV3101. (1)Type tobacco is infected by leaf disc method to transgenic tobacco selected on solid medium containing hygromycin. Total DNA of positive plants as template, with non transgenic tobacco as negative control, recombinant plasmid as positive control, amplifying GUS sequence to identify transgenic plants. The result shows stripe amplified by PCR is exactly the same as GUS sequence and the non-transgenic tobacco does not amplify stripes. (2)Type Arabidopsis is infected by agrobacterium GV3101 and transgenic arabidopsis is successfully identified by sowing seeds of T1 generation on solid medium containing hygromycin.