Expression of Phytase gene in transgenic maize with seed-specific promoter 27-kDa γ Zein and constitutive promoter CaMV 35S

Phytase enzymes are applied to animal feed to help animals absorb more nutrients. The use of feed raw materials containing phytase enzymes is expected to reduce the cost of animal feed production. Efforts to increase the phytase content in maize were carried out by improving genetics, in the way of assembling transgenic plants containing high phytase content. The 27-kDa γ Zein promoter is a specific promoter that expresses genes in caryopsis, and promoter CaMV 35S is a constitutive promoter that controls gene expression in all tissues and generally does not depend on the growth phase. Transgenic maize was transformed using Agrobacterium tumefacien infection method on maize B104. The reverse transcriptase polymerase chain reaction (RT-PCR) approach was used to examine the expression of phytase genes in leaves, roots, and caryopsis was done 10, 20, and 30 days after pollination (DAP). The phytase enzyme activity test was also carried out by using the colorimetric phosphomolybdate analysis method to see the phytase enzyme activity in unit µg-1. The results showed that the phytase gene in transgenic plants with the 27-kDa γ Zein promoter was highly expressed in maize caryopsis, but in line Z6.10 was also expressed in leaves, while in the CaMV 35S promoter the phytase gene was only expressed on the leaves. Phytase enzyme activity showed that transgenic maize was higher than non-transgenic maize.

Cloning of Bacillus subtilis phytase gene construct in Escherichia coli

Background and Objectives: Phytase has a hydrolysis function of phytic acid, which yields inorganic phosphate. Bacillus species can produce thermostable alkaline phytase. The aim of this study was to isolate and clone a Phytase gene (Phy) from Bacillus subtilis in Escherichia coli. Materials and Methods: In this study, the extracellular PhyC gene was isolated from Bacillus subtilis Phytase C. After purification of the bands, DNA fragment of Phy gene was cloned by T/A cloning technique, and the clone was transformed into Escherichia coli. Afterward, the pGEM-Phy was transferred into E. coli Top-10 strain and the recombinants were plated on LB agar containing 100 µg/ml ampicillin. The colonization of 1171 bp of gene Phytase C was confirmed by PCR. The presence of gene-targeting in vector was confirmed with enzymatic digestion by XhoI and XbaI restriction enzymes. Results: The Phytase gene was successfully cloned in E. coli. The result of cloning of 1171 bp Phytase gene was confirmed by PCR assay. Conclusion: Our impression of this article is that several methods, such as using along with microbial, plant phytase reproduction, or low-phytic acid corn may be the better way from a single phytase.

Increased Nodular P Level Induced by Intercropping Stimulated Nodulation in Soybean Under Phosphorus Deficiency

Low P availability is a vital constraint for nodulation and efficient N2 fixation of legume, including soybean. To elucidate the mechanisms involved in nodule adaption to low P availability under legume/cereal intercropping systems, two experiments consisting of three cropping patterns (monocropped soybean, monocropped maize, soybean/maize intercropping) were studied under both sufficient-and deficient-P levels. Our results demonstrated that intercropped soybean with maize showed a higher nodulation and N2 fixation efficiency under low P availability than monocropped soybean as evidenced by improvement in the number, dry weight and nitrogenase activity of nodules. These differences might be attributed to increase in P level in intercropping-induced nodules under low P supply, which was caused by the elevated activities of phytase and acid phosphatases in intercropping-induced nodules. Additionally, the enhanced expression of phytase gene in nodules supplied with deficient P level coincided with an increase in phytase and acid phosphatase activities. Our results revealed a mechanism for how intercropped maize stimulated nodulation and N2 fixation of soybean under P deficient environments, where enhanced synthesis of phytase and acid phosphatases in intercropping-induced nodules, and stimulated nodulation and N2 fixation.

Construction of a new plant expression vector and the development of maize germplasm expressing the Aspergillus ficuum phytase gene PhyA2

Phytases, which belong to a special category of orthophosphoric monoester phosphohydrolases, degrade inositol hexaphosphate to produce lower-grade inositol phosphate derivatives and inorganic phosphate. Thus, phytases may improve phosphorus utilization, eliminate the anti-nutrient properties of phytic acid, and mitigate environmental pollution due to phosphorus contamination. In this study, we constructed a new root-specific expression vector by inserting the Aspergillus ficuum phytase gene PhyA2 into pCAMBIA3301-ZmGLU1P-Nos. The subsequent molecular analysis confirmed that six T4 generation transgenic plants carried and expressed PhyA2. A quantitative real-time PCR analysis indicated PhyA2 was highly expressed in the transgenic roots. Additionally, the phytase activity was 10.9-fold higher in the transgenic roots (peak activity of 5.432 U/g) than in the control roots. Moreover, compared with the control rhizosphere, the organic phosphorus content in the rhizosphere of the transgenic plants decreased significantly (by up to 5.21 mg/kg). An agronomic trait analysis indicated that PhyA2 expression can increase maize seed weight by up to 25.8 g. Therefore, the integration of PhyA2 into the maize genome can enhance the ability of maize plants to use the phosphorus compounds in soil, while also improving the plant growth status and increasing the seed yield.