| Pcpg (Phytophthora cinnamomi polygalacturonase) 1, Pcpg2, Pcpg4, Pcpg6, Pcpg7, Pcpg9, Pcpgl0, Pcpg12, Pcpg16, Pcpg17, Pcpg19, Ppgip (pear polygalacturonase-inhibiting proteins) and Anpg (Aspergillus niger polygalacturonase)I genes or/and their exons were cloned, sequenced and analysed, ligated, and their expression vectors were constructed based on the studying on the PCR composition and its reacted condition of the genes and exons. Then, the target genes were transformed to a yeast (Saccharaomyces cerevisiae) line of W303-1B. After that, the expression of target genes was analysed by application the techniques of Western blotting and method of plate assay polygalacturonase activity. Finally, the interaction of the gene expression between pg genes and Apgip (Arabidopsis polygalacturonase-inhibiting proteins) gene, or Tpgip (tomato polygalacturo-nase-inhibiting proteins) gene, or Ppgip gene was initially studied. Besides, the time and method of inducing the expression of pg genes was tested, and the techniques and method of colony PCR for Esherichia coli and Saccharomyces cerevisiae were standardized. The results were as follow.1.The yeast expression vectors of Pcpg1, Pcpg2, Pcpg4, Pcpg6, Pcpg7 Pcpg9, Pcpg10, Pcpg12, Pcpg16, Pcpg17, Pcpg19, Ppgip and Anpgl genes were constructed based on a yeast vector of pYES2. The expression vectors contain the sequence of GAL1 promoter (galactose inducible promoter), ampicillin resistant gene, URA3 gene, 2 micron origin of replication, the a factor pre-pro , Kex2 cleavage site and 3×HA (hemagglutinin) tag. The a factor pre-pro sequence is a leader sequence important for proper secretion of the protein. This sequence is cleaved from the protein by signal peptidase at the signal peptide cleavage site in the lumen of the rough endoplasmic reticulum and by Kex2 protease at the Kex2 cleavage site in the late Golgi apparatus before it is secreted. The 3× HA tag is an epitope tag introduced into the protein to enable detection of the protein using antibodies against the 3× HA tag. Colony PCR and enzyme cleavage analysis indicated that the construction vectors were perfect, and they can be used in yeast transformation.2. The optimization PCR condition of Pcpgl, Pcpgl, Pcpg4, Pcpg7, Pcpg10, Pcpgl9 and Ppgip genes, as well asAnpglE1, AnpglE2, AnpgIE3, Pcpg6E1, Pcpg6E2, Pcpg9E1, Pcpg9E2, PcpgUE1, Pcpgl2E2, Pcpgl6E1, Pcpgl6E2, PcpgllE2 and Pcpg17E3 exons were obtained by sequencing analysis.3. Sequencing analysis and the results of gene expression showed that the size ofPcpgl, Pcpg2, Pcpg4, Pcpg6, Pcpg7, Pcpg9, Pcpg10, Pcpg12, Pcpgl6, Pcpg17, Pcpg19 and Ppgip genes was 1011bp, 1011bp, 1011bp, 1017bp, 1170bp, 1059bp, 1290bp, 1293bp, 996bp, 996bp, 1146bp and 1026bp respectively.4. Further clarify the sequence of Pcpg1, Pcpg2, Pcpg4, Pcpg6, Pcpg7, Pcpg9, Pcpg10, Pcpgl2, Pcpg16, Pcpg17, Pcpg19 and Ppgip genes by comparing the sequencing results of cloned Pcpg genes and Ppgip gene with the original machine reading results of genomic library. Make corrections of the original machine reading results of genomic library, such as, replacing alanine (GCC) of 390th amino acid in Pcpg10 to glycine (GGC), and replacing lencme (TTG) of 219th amino acid in Pcpgl 2 to Phenylalanime (GGC).5. The transgenic yeast lines of Pcpg1, Pcpg2, Pcpg4, Pcpg6, Pcpg7, Pcpg9, Pcpg10, Pcpg12, Pcpg16, Pcpg11, Pcpg19 and Ppgip genes were obtained respectively. The PG activity assay showed that the yeast lines of Pcpg2, Pcpg4, Pcpg6, Pcpg7, Pcpg9, Pcpg10, Pcpgl2 and Pcpgll genes expressed PG activity and they was function genes, that the yeast lines of Pcpg1, Pcpgl 6 and Pcpgl 9 did not express PG activity and they was nonfunctional genes. The results of Western blotting indicated that Pcpg1, Pcpg16 and Pcpgl 9 genes could guide to synthetise the secretion protein which was nonfunctional. Different pg genes expressed different PG activity. The secretion PG activity of transformed yeast lines of Pcpgl, Pcpg1 and Pcpg10 was the strongest, and that of AnpgI Pcpg4, Pcpg12 and Pcpgll was more st...
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