利用子叶下胚轴为外植体，通过农杆菌(Agrobacterium tumefaciens) Ti质粒介导遗传转化途径，建立了高效表达黑曲霉PhyA基因的白三叶草转基因系。PCR和Southern印迹结果表明，PhyA基因已整合到转基因植株的基因组中。Northern印迹结果表明，部分转基因系中的PhyA基因具有高表达水平。此外，PhyA基因表达的植酸酶在Patatin信号肽的引导下具有分泌至细胞间隙和根际的能力。与未转基因对照相比，在以植酸盐为唯一磷素供源的条件下，高表达PhyA的转基因系植株的磷素累积量、鲜重和干重显著增加。证明外源转化黑曲霉PhyA基因能显著增强白三叶草利用有机态磷的能力。
The open reading frame (ORF) of phytase (PhyA) and cDNA part of patatin signal peptide were PCR amplified separately with DNA of Aspergillus niger and potato. The binary expression vector fused PhyA ORF and potato patatin signal peptide was constructed. Using the cotyledon of white clover as explants, the transgenic white clover lines ectopic expression of the PhyA gene were established based on Agrobacterium tumefaciens-mediated transformation method. The wild type plants without transformed (CK) and ten randomly selected transgenic lines were used for PCR amplification analysis to detect the target gene in the transgenic lines. It was found that the tested transgenic lines all showed positive PCR. The transgenic line 1 to line 4 were used for further Southern blot and Northern blot analysis. The results indicated that the genome of above four lines all contained low copies of PhyA. The line 1 and line 3 had higher levels of PhyA expression, indicating that it is effective to conduct genetic transformation of ectopic target genes in white clover by using agrobacterium tumafeciens-mediated method.The line 1 and line 3 with higher level of PhyA expression were used to assay the phytase activities in root and its intercellular space. When the phytate was the only phosphorus source, the phytase activities in root in line 1 and line 3 were 31.43% and 44.76% higher than those in control plants (CK), respectively. Meanwhile, the phytase activities in the intercellular space of root in line 1 and line 3 were 3.3-fold and 5.12-fold higher than those in CK, respectively. Therefore, the PhyA products in line 1 and line 3 could be secreted into the intercellular space of root by the direction of patatin signal peptide. Similarly, under the condition with phytate as the only source of phosphorus, the phosphorus concentration of plants, the accumulative phosphorus amount per plant, plant fresh weight and plant dry weight were all much higher in line 1 and line 3 than in CK. Among them, for line 1 and line 3 the phosphorus concentration of plant was 30.23% and 34.88% higher than CK, the accumulative phosphorus amount per plant was 2.90-fold and 3.54-fold, the plant fresh weight was 2.25-fold and 2.79-fold and the plant dry weight was 2.22-folf and 2.79-fold, respectively compared with the CK. Thus, it was clearly shown that ectopic expression of Aspergillus niger PhyA gene can significantly increase the ability for white clover to utilize organic phosphate under Pi-deficient condition.
全 文 ：