Study On Soil Phosphorus Uptake And Utilization By Tobacco Plants Overexpressing Malate Dehydrogenase And Citrate Synthase From Penicillium Oxalicum

Phosphorus deficiency is considered to be one of the major limiting factors to the improvement of field crops, due to the strong adsorption, low solubility, difficult mobility of phosphorus, as well as some other reasons. Worldwide, acidic and alkaline soils make up about 70% of the arable soil. Most of the phosphorus in these soils exists as insoluble phosphate, which is largely unavailable to plant. In order to enhance the efficiency of phosphorus usage and ensure plant productivity, nearly 30 million tons of phosphorus fertilizer are applied worldwide every year, of which, up to 80% can’t be utilized by plants because of adsorption, precipitation or conversion to organic forms. So, enhancing the ability of the plant to use soil phosphorus will play an import role in improving crop production and agro-ecological environment. There has been research showing that altering the metabolism of organic acid in plants could increase the secretion of organic acid, a potential mechanism that could enhance the utilization of insoluble phosphate by these plants.In this study, the genes of two key enzymes, citrate synthase (Cit) and malate dehydrogenase (MDH), that are involved in the oganic aicd metabolic pathway were isolated from Penicillium oxalicum, and the homology and secondary structures of putative proteins were analyzed. The two genes were also overexpressed in E.coli and tobacco, and the growth response of transformants to phosphorus deficiency was studied. The research contents and the results obtained are stated in details as follows:(1) The full-length genes encoding malate dehydrogenase (Po-mMDH) and citrate synthase (Po-mCit) of P. oxalicum were cloned by RT-PCR and RACE techniques. The full-length of Po-mMDH cDNA (GenBank accession no. FJ550602) was 1284-bp long and it contained an ORF of 1023 bp, which encodes a polypeptide of 340 aa. The nucleotide sequence had an untranslated regions of 45 bp at the 5’-end, and 216 bp at the 3’-end. The deduced transcript of Po-mMDH had 85% identity with the mMDHs of Aspergillus terreus and Aspergillus fumigatus. Po-mMDH also contained a 23-aa mitochondrial-targeting signal peptide at the N-terminus. The full-length of Po-mCit cDNA (GenBank accession no. GQ981487) was 1716-bp long, containing an ORF of 1416 bp, which encodes a polypeptide of 471 aa. The nucleotide sequence had an untranslated regions of 96 bp and 205 bp at the 5’-and 3’-ends, respectively. The deduced transcript of Po-mCit had 85% identity with the mCit of Aspergillus nidulans and Aspergillus niger. Po-mCit also had a 31-aa mitochondrial-targeting signal peptide at the N-terminus.(2) The mature genes of Po-mMDH and Po-mCit were cloned into the vector pET32a, and then transformed into E. coli strain BL21(DE3) to study their expressions in prokaryotic cell. Expressions of soluble proteins were obtained in both cases. BL21(DE3) transformed with Po-mMDH showed high MDH activity (48.7μmol/mg protein), about 5-fold higher than control. When grown in media with tricalcium phosphate as the only source of phosphorus, the transformant secreted more malate, lactate, acetate, citrate, oxalate, at about 2.7-、3.7-、1.9-, 2.3- and 9.1- folds, respectively, higher than control. Soluble phosphorus content of the transformant (134.8 mg/L) also increased significantly over control. BL21(DE3) transformed with Po-mCit showed about 11-fold increase in Cit activity. When grown in medium with tricalcium phosphate as the unique source of phosphorus, the transformant secreted about 2.3-,1.6-,2.9-,3.5- and 7.6- folds more citrate, malate, lactate, acetate and oxalate, respectively, than control. The soluble phosphorus content of the transformant was significantly increased. These results show that expressions of P. oxalicum mMDH and mCit genes in E. coli could enhance organic aicds secretion and improve the phosphate solubilizing ability of the host cells.(3) The ORF of Po-mMDH was cloned into the plasmid pBI121, and transferred into tobacco via Agrobacterium-madiated method. Positive transformants were verified by PCR, Sourthern blot and RT-PCR analysis. Three T2 progenies of homozygous plants (M1, M2 and M3) each harboring a single copy of the transgenic gene were selected for further analysis to the level of MDH expression, root malate exudate, and the ability of the plant to utilize insoluble form of phosphorus. All transgenic lines showed significantly higher level of MDH activity compared to that of wild type. Malate content in the root exudation of the transgenic lines induced in response to phosphorus deficiency was 1.3- to 2.9-fold higher than that of wild type. Among these transgenic lines, one line (M1) showed the highest level of MDH activity and malate exudate. M1 also showed a significant increase in growth over wild type, with 2.5-,2.3- and 2.3-fold increases in biomasses when grown in Al-phosphate, Fe-phosphate and Ca-phosphate containing media, respectively. M1 also had better phosphorus uptake compared to wild type, with 3.9-,3.4- and 3.2-fold increases in total phosphorus content when grown in Al-phosphate, Fe-phosphate and Ca-phosphate media, respectively.(4) The ORF of Po-mCit was cloned into the plasmid pCAMBIA1301 and transferred into tobacco via Agrobacterium-madiated method. The transformed tobacco plants were verified by PCR, Sourthern blot and RT-PCR analysis. Four T2 progenies of homozygous plants (C1, C2, C3 and C4) each harboring a single copy of the transgenic gene were selected and the level of Cit expression, the ability of the plant to utilize different forms of phosphorus and growth were investigated. The levels of Cit activity in the transgenic lines were significantly increased compared to that of wild type. Cit activity of these transgenic plants determined under different phosphorus conditions (N-P, Al-P, K-P) all produced the highest level of Cit activity when grown under insoluble phosphorus condition (Al-P). The concentration of citrate exuded by the transgenic lines was also significantly increased, with C1, C2, C3 and C4 reaching about 1.6-,2.7-,2.5-and 2.3-fold, respectively, higher than wild type. Under insoluble phosphorus condition (A1-P), significant improvements in plant growth and phosphorus utilization were observed for the transgenics lines. The transgenic tobaccos accumulated more shoot biomass and root biomass. The total phosphorus concentration of transgenic lines C1-C4 was also significantly increased, reaching about 1.6-,2.4-,1.9-and 1.8-fold higher over that of wild type.The results presented in this thesis indicated that by altering the organic acid metabolism of a plant, e.g., through over expressions of malate dehydrogenase and citrate synthase, the ability of the transgenic plant to utilize insoluble form of phosphorus could be enhanced, which may enable the plant to grow in soil where phosphorus may not be so readily available.