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.


Introduction
As one of the three most important crops, maize is widely used, including as food, feed, and industrial raw materials (Wu et al. 2014). Early maize producers used traditional crop breeding techniques to develop and cultivate many excellent maize varieties. Recently, genetic engineering and transgenic techniques have been applied to transfer genes associated with specific functions into maize inbred lines to enhance crop quality and specific plant traits. Advances in molecular breeding research have provided researchers with new options for the genetic improvement of maize.
Phosphorus is one of the nutrients essential for maize growth and development. Soil organic phosphorus is an important source of phosphorus nutrients for maize (Sareh et al. 2018). Organic phosphorus represents approximately 20-80% of the total phosphorus content in soil, with more than 50% of the organic phosphorus existing as phytate phosphorus, which is difficult for plants to absorb and use (Enqing et al. 2018). Phytate phosphorus is also an important form of phosphorus in maize plants, especially in seeds, wherein it accounts for about 70% of the total phosphorus content. However, the digestive tract of monogastric animals lacks enzymes to hydrolyze phytate phosphorus, resulting in its excretion into the environment in feces. This not only decreases the phosphorus utilization by animals, it also increases environmental pollution due to organic phosphorus (Yan 2012). Additionally, phytate phosphorus can chelate other metal ions to form compounds that are relatively insoluble. Therefore, phytate phosphorus is also known as an anti-nutrient factor. Phytases are a class of enzymes that can catalyze the hydrolysis of phytate phosphorus and release inorganic phosphate and inositol that can be passively absorbed and used by plants (Valeeva et al. 2018).
In this study, the PhyA2 gene, which encodes a phytase, was cloned from Aspergillus ficuum and used to construct a new plant expression vector containing a root-specific promoter. By applying genetic engineering technology, the recombinant plasmid was inserted into a maize inbred line. The root-specific expression of PhyA2 increased the phytase content and activity to improve the degradation of the organic phosphorus in soil, enabling the transgenic maize plants to absorb more phosphorus and efficiently degrade phytic acid salt. The results of this study may be useful for producing new maize germplasm that can efficiently use phosphorus to increase the crop yield.

Materials and methods
Experimental strains, plasmid vector, and plant receptor material Aspergillus ficuum was purchased from the Institute of Microbiology, Chinese Academy of Sciences.
Escherichia coli DH5a and Agrobacterium tumefaciens EHA105 cells, the modified plant expression vector pCAMBIA3301-ZmGLU1P-Nos, and the germinal callus cells of maize inbred line GSH9901 were provided by the Key Laboratory of Crop Molecular Breeding in Jilin Province, China.
Preparation of the maize embryo and induction of the embryogenic callus Young maize ears (10-13 days old) were selected. The outer bract was removed, leaving a layer of leaves, after which the maize ears were washed with 75% ethanol for 15 min. The leaves were peeled off and the palpus was removed before the maize ears were disinfected with 0.5% NaClO solution for 10 min and then rinsed with sterile water 3-4 times. In an ultraclean workbench, a scalpel was used to cut the maize kernels. After removing the seed coat and endosperm, the young embryo was transferred to N6 medium for a 15-day incubation at 25°C in darkness.

Cloning of PhyA2 from Aspergillus ficuum
The A. ficuum PhyA sequence (AF537344.1) available in the GenBank database was used to design genespecific primers (S and AS) with the Primer Premier 5.0 program. The primers included XbaI and HindIII restriction enzyme sites. Details regarding the primer sequences are provided in Table 1.
Total RNA was extracted from A. ficuum using a commercial RNAiso plus extraction kit (Bao Bioengineering (Dalian) Co., Ltd., Economic and Technological Development Zone, Dalian City, Liaoning Province, China) and then reverse transcribed to cDNA. The cDNA was used as the template for a PCR amplification of the target gene (PhyA2) with primers S and AS. The PCR conditions are listed in Table 2. The PCR product was analyzed by agarose gel electrophoresis. After purifying the amplified PhyA2 fragment from the gel, it was inserted into the pMD18-T vector. The resulting pMD18-T-PhyA2 recombinant plasmid was sequenced to confirm the accuracy of the PhyA2 sequence.

Construction of a new plant expression vector
The pMD18-T-PhyA2 plasmid and the modified plant expression vector pCAMBIA3301-ZmGLU1P-Nos were digested with XbaI and HindIII and then analyzed by agarose gel electrophoresis. The PhyA2 gene fragment and the linearized plant expression vector sequence were recovered and ligated with T 4 DNA ligase. The resulting new plant expression vector was analyzed by a PCR amplification and a restriction enzyme digestion to confirm it was constructed correctly. The process involved in constructing the new plant expression vector is presented in Fig. 1.

Transformation of maize with the plant expression vector
The PhyA2 gene was inserted into maize inbred line GSH9901 embryonic calli via A. tumefaciens-mediated transformation. The optimized infection and transformation conditions were as follows: 25-30 min for the A. tumefaciens infection of calli; the bacterial culture was used after reaching an optical density at 600 nm (OD 600 ) of 0.6-0.7; and 2-3 days for the co-culture required for the A. tumefaciensmediated transformation. After screening for herbicide (Glufosinate) resistance, calli were differentiated. When the herbicide-resistant calli on the differentiation medium 1 grew to 3-5 cm with 3-4 leaves, the seedlings were transferred to the rooting medium. Approximately 15-25 days later, 4-6 robust primary roots and many fibrous roots were detected. The seedlings were incubated at 23-26°C for 2-3 days, after which they were transplanted to pots containing sterilized soil.

Agrobacterium tumefaciens culturing
Agar-solidified YEB medium supplemented with 50 mg/L Kan was inoculated with A. tumefaciens cells containing the recombinant plant expression vector carrying PhyA2. The culture was incubated at 28°C for 2 days. An individual colony was added to a test tube containing 5 mL YEP/Kan medium. The culture was oscillated (180 rpm) overnight at 28°C. A 3-mL aliquot of the overnight culture was added to 50 mL YEP/Kan medium, after which the culture was incubated at 28°C, with shaking at 220 rpm. When the OD 600 of the bacterial culture reached approximately 0.7, the culture was centrifuged at 5000 rcf for 20 min at room temperature to collect the bacteria. The bacterial cells were resuspended in the same volume of MS liquid medium.

Callus transformation and co-culturing
The callus was added to 50 mL infection solution in a sterile culture bottle, which was then shaken for 6 min. The infected callus was removed from the bottle, placed on sterile filter paper to absorb the excess infection solution, and then transferred to the coculture medium for a 2-day incubation in darkness.

Screening and culturing
After the co-culture step, the embryogenic callus exhibiting healthy growth (e.g., soft and pale yellow) was transferred to the screening medium for an incubation at 28°C under light. The medium was refreshed every 15 days.

Rooting
After the adventitious buds grew to 2-3 cm, they were transferred to the rooting medium. After 1 week, adventitious roots were detected, enabling the transfer of plants to soil.

PCR analysis of transgenic plants
Genomic DNA was extracted from herbicide-resistant T 3 generation maize plants with a NuClean Plant Genomic DNA kit (Changchun Feikai Biological Co., Ltd., Economic Development Zone, Changchun City, Jilin Province). The maize genomic DNA was used as a template for a PCR with primers S and AS. The plant expression vector carrying PhyA2 was used as the positive control, whereas the genomic DNA from untransformed maize plants served as the negative control. The PCR conditions are listed in Table 2. The PCR products were analyzed by agarose gel electrophoresis. The T 3 generation maize plants that were confirmed by PCR to be transgenic were self-pollinated to produce seeds. The T 3 generation seeds were germinated to obtain T 4 generation transgenic maize plants, which were verified by PCR.

Southern hybridization analysis of transgenic plants
Large quantities of genomic DNA were extracted from the T 4 generation transgenic maize plants and untransformed maize plants (positive control) according to a modified CTAB method (Abdel and Osman 2017). A Southern blot analysis was completed with purified PhyA2 as the probe as well as the plant expression vector carrying PhyA2 and the genomic DNA from untransformed maize plants as the positive and negative controls, respectively. Specifically, 10-15 lg maize genomic DNA was digested with HindIII for 16 h. The digested DNA was separated in a 0.8% agarose gel and then transferred to a nitrocellulose membrane in a 209 SSC solution. The membrane was then incubated at 80°C for 2 h, after which the DNA was hybridized with the probe.

Reverse transcription PCR analysis of transgenic plants
Total RNA was extracted from the roots of transgenic maize plants and reverse transcribed to cDNA. Total RNA was also extracted from the roots of untransformed maize plants and reverse transcribed (negative control). The cDNA samples for the transgenic and control maize plants were used as the templates for a PCR amplification of PhyA2 with primers S and AS ( Table 1). The maize EF-1a gene, which was used as the internal reference control, was amplified by PCR with primers P1 and P2 to produce a 213-bp fragment ( Table 3). The PCR conditions are listed in Table 2.

Quantitative real-time PCR analysis of transgenic plants
Total RNA was extracted from the roots, stems, and leaves of untransformed (control) and transgenic maize plants and reverse transcribed to produce cDNA for a quantitative real-time (qRT)-PCR analysis of PhyA2 expression. The maize beta-actin gene (Gen-Bank accession number: nm_001252731.2) was selected as the internal reference control. Primers QFACT and QRACT (Table 4) were designed based on the PhyA2 sequence. The qRT-PCR analysis was completed with the SYBR Green fluorescent dye. The reaction conditions are listed in Table 5. Each sample was analyzed with three replicates. The relative PhyA2 expression levels in the different maize tissues were calculated according to the 2 -DDCt method (Yueai et al. 2014).

Preparation of the phosphorus concentration standard curve
A phosphorus concentration standard curve was prepared as previously described (Safety certificate 2009). Additionally, a published method for analyzing  (Zhang 2009) was modified to examine the T 4 generation transgenic maize plants.
Specifically, 2 g root tissue was ground to a homogenate, which was then mixed with 8.0 mL extraction solution (0.25 mol/L sodium acetate, 0.05% Triton X-100, and 0.05% bovine serum Fig. 1 The process of the construction of plant expression vector albumin). The resulting solution underwent an ultrasonication for 15 min at 37°C in a water bath, after which it was centrifuged at 12,000 rcf.

Determination of the phytase activity in transgenic plant root tissue
Phytase activity was assessed in three parallel experiments as previously described (Bouchra et al. 2018;Bekalu et al. 2017). The root tissue phytase activity [i.e., 1 unit (U)] was defined as the amount of enzyme required to release 1 lmol inorganic phosphorus from 0.10 mmol/L sodium phytate solution in 1 min at 37°C and pH 5.5. The enzyme activity was expressed as U/g.

Analysis of the phosphorus in the rhizosphere of transgenic plants
The T 4 generation transgenic maize plants verified by the Southern blot analysis were grown in soil for an analysis of the organic phosphorus content in the rhizosphere (i.e., soil up to 20 cm deep and within 5 cm of the main root). Soil samples collected before the transgenic maize were planted were used as controls.

Analysis of the agronomic characteristics of transgenic maize plants
Three replicates of the T 3 and T 4 generation transgenic maize plants were grown in an experimental field at Jilin Agricultural University to evaluate the main agronomic traits. At maturity, plant height and other yield-related characteristics were analyzed.

Construction and detection of a new plant expression vector
The results of an agarose gel electrophoresis (Fig. 2) revealed the PhyA2 fragment amplified by PCR was approximately 1400 bp long, which was the expected size. The new plant expression vector was digested with XbaI and HindIII and the resulting fragments were examined in an agarose gel (Fig. 3). Two fragments were obtained following the enzymatic digestion, one of which was about 1400 bp, as expected for PhyA2. Thus, the PCR and restriction enzyme digestion confirmed that the new plant expression vector (pCAMBIA3301-ZmGLU1P-PhyA2-Nos) was constructed correctly.
Agrobacterium tumefaciens-mediated genetic transformation of the maize callus An A. tumefaciens-mediated method was used to transform a maize inbred line with PhyA2. Six herbicide-resistant T 4 generation maize plants were obtained. The specific transformation process is presented in Fig. 4.

PCR detection of PhyA2 in transgenic maize plants
Genomic DNA was extracted from the leaves of transgenic maize plants for a PCR analysis to confirm the insertion of PhyA2. The PCR product was examined by 1% agarose gel electrophoresis. The    . 7) revealed single-copy hybridization bands for two plants, whereas bands were undetectable for the untransformed control plants. Thus, the integration of PhyA2 into the maize genome was confirmed.

Reverse transcription PCR identification of transgenic maize plants
The PhyA2 and EF-1a gene sequences were amplified by PCR using cDNA prepared from the total RNA extracted from maize roots. The expected PhyA2 and EF-1a sequences were amplified for the transgenic plants, whereas only the EF-1a sequence was amplified for the untransformed plants (Fig. 8). Thus, the reverse transcription PCR results confirmed that PhyA2 was expressed in the roots of the transgenic maize plants.

Quantitative real-time PCR detection of transgenic lines
A qRT-PCR assay was performed to analyze the PhyA2 expression levels in the young leaves, roots, and stems of T 4 generation transgenic plants verified by PCR and Southern blotting. The PhyA2 expression levels were calculated relative to the expression of the maize beta-actin gene. The qRT-PCR data indicated that PhyA2 was more highly expressed in the transgenic maize plants than in the control plants (Fig. 9). Specifically, the PhyA2 expression level was 3.75times higher in the transgenic maize roots than in the control roots. Additionally, the mean PhyA2 expression levels in the transgenic stems and leaves were respectively 1.42-times and 1.84-times higher than in the corresponding control tissues. Thus, PhyA2 was specifically expressed in the transgenic maize roots.

Phosphorus concentration standard curve
A phosphorus concentration standard curve was prepared with the phosphorus concentration on the abscissa and the absorbance on the ordinate (Fig. 10). The linear regression equation was y = 1.026x ? 0.0053, R 2 = 0.9996, reflecting the relatively linear relationship between the phosphorus concentration and absorbance.

Phytase activity analysis of transgenic maize plants
The phytase activity in the T 4 transgenic maize roots was compared with that in the roots of untransformed plants. The phytase activity was significantly (P \ 0.05) higher in the transgenic roots than in the control roots (Table 6). On average, the phytase activity was 10.9-fold higher in the transgenic roots than in the control roots. The increased phytase activity of the transgenic roots ranged from 17.6-fold (T4-3-13) and 4.8-fold (T4-33-12) higher than that in the control roots. These results implied that PhyA2 was correctly incorporated into the genome and was being expressed in the roots of the transgenic maize plants. Moreover, the phytase produced in the transgenic plants appeared to be highly active.  (Table 7). Thus, the expression of a phytase gene in maize roots appears to affect the soil organic phosphorus content.

Phenological growth stages of transgenic maize
There were no significant differences in the phenological growth stages of the transgenic (e.g., 35-15, 36-10, and 44-18) and control maize plants (Table 8). However, the tasseling stage occurred 3 days earlier in the transgenic plants than in the control plants.
Additionally, the 6-13 transgenic plants reached maturity 4 days later than the controls.

Analysis of the plant height and seed weight of the transgenic maize
The ear length and thickness were similar in the transgenic and control plants (Table 9), but the effects of natural factors on the transgenic maize plants remain to be assessed. Additionally, although the height of individual transgenic maize plants differed from the mean height of control plants by 2-3 cm, this difference was not significant. Accordingly, PhyA2 expression does not appear to affect maize plant height. Moreover, the mean 100-grain weights of the 6-13 and 13-7 transgenic plants were lower than that of the control plants. In contrast, the mean 100-grain weight of the other transgenic maize plants was 0.56 g higher than that of the control plants. Therefore, PhyA2 expression may influence maize seed weights.

Discussion
Phytase was initially identified in 1915, and its considerable ability to degrade phytic acid was subsequently confirmed by Anderson (1941). The rapid development of biotechnology has enabled researchers to investigate the effects of the ectopic expression of phytase genes in transgenic plants. For example, Abul et al. (1999) isolated a PhyA gene from A. ficuum, and successfully expressed it as a recombinant gene in tobacco leaves. Although the molecular weight and optimal pH was lower for the expressed product than for the natural phytase, the biological characteristics were otherwise identical. The PhyA2 gene was successfully cloned from A. ficuum cDNA in this study. In terms of molecular biology, a vector can be used to transfer a target gene into the receptor material and subsequently control the expression of the target gene (Chen 1994). A good plant expression vector is important for the successful transformation of plants with exogenous DNA material. Different types of regulatory elements in plant expression vectors can directly affect the expression of recombinant phytase T 4 -3-13 comes from T 3 -7-1 of T 3 generation plants. T 4 -4-20 comes from T 3 -5-2 of T 3 generation plants. T 4 -5-4 comes from T 3 -8-3 plants of T 3 generation. T 4 -6-13 comes from T 3 -9-4 of T 3 generation plants. T 4 -13-7 and T 4 -13-11 came from the same T 3 plant T 3 -16-7. T 4 -14-1 and T 4 -14-3 came from the same T 3 plant T 3 -20-3. T 4 -16-13 comes from T 3 -22-6 of T 3 generation plants. T 4 -28-19 comes from T 3 -23-4 of T3 generation plants. T 4 -33-12 comes from T 3 -27-9 of T 3 generation plants. The 5% level is significantly indicated by lowercase letters, and the 1% level is significantly indicated by uppercase letters Note: Each set of data in the graph is the mean ± standard deviation of three replicates of three sites in each plant. ''*, **'' represents the difference in significance between the transgenic positive plants and the untransformed plants, respectively. ''*'' stands for 0.01 \ P \ 0.05 for significant difference; ''**'' stands for 0.001 \ P \ 0.01 for significant difference genes in plants. For example, the ZmGLU1 promoter may be used for the root-specific expression of a transgene. In the current study, PhyA2 was inserted into a plant expression vector containing the ZmGLU1 promoter for the root-specific expression of the phytase gene. This study was similar to that conducted by Cui (2007). Our gene expression analyses confirmed that the inserted PhyA2 gene was expressed in the T 3 and T 4 generation transgenic maize plants. Additionally, the PhyA2 expression level in the transgenic maize plants was higher in the roots than in the leaves. The observed phytase activities in this study were consistent with those determined by Zhang et al. (2010). Specifically, the phytase produced in the transgenic maize roots was able to degrade sodium phytate; however, the relatively low phytase activity in this study may have been due to the instability of PhyA2, resulting in impure transgenic maize plants. Li et al. (2009) reported similar results for their investigation of transgenic soybean plants expressing a phytase gene. William et al. (1990) were the first to produce fertile transgenic maize plants, making it possible to improve maize traits via methods commonly used for biotechnology research. Our analysis of soil phosphorus levels revealed a decrease in the organophosphorus content in the rhizosphere of transgenic plants relative to the content in the control rhizosphere. This decrease may reflect the degradation and uptake of organophosphorus by the transgenic plants expressing PhyA2; however, the phosphorus use efficiency was relatively low. Song et al. (2008) explored the effects of phytase on soil organophosphorus contents. Their results indicated that increases in the abundance of active or moderately active organophosphorus compounds in the soil may be related to increases in phosphatase activities. Moreover, the phosphatase activity may decrease with increases in moderately or highly stable organophosphorus compounds in the soil.
In this study, analyses of phenological growth stages revealed a lack of significant differences between the PhyA2-expressing transgenic maize plants and the control plants. Additionally, there were no significant differences in the physiological indices of the transgenic and control maize plants, possibly because the plants were not exposed to adverse environmental conditions. Furthermore, PhyA2 However, there were some differences in the mean 100-grain weight, with higher weights in the transgenic plants than in the controls, except for 6-13 and 13-7. These findings suggest that maize plants expressing a phytase gene may be able to take up organic phosphorus from the soil and transfer it to the developing ears.
In summary, compared with the control maize inbred line GSH9901, the introduction of PhyA2 into maize can positively affect the soil phosphorus use efficiency, plant growth state, and seed yield. Therefore, PhyA2 may be useful for improving maize yield traits and phosphorus use efficiency.

Compliance with ethical standards
Conflict of interest The authors declare that they have no competing interest.
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