Cloning, Expression And Characterization Of Phytase PhyC Gene And Studies On Transformation In Mulberry

In China, with its long period of traditional of practicing sericulture and mulberry culture, there have been a large number of indigenous cultivars; it was one of the great contributions to human civilization. As an important economic tree species in China, mulberry Morus L. is a kind of perennial woody plant with twin cotyledon and its' leaf is the main natural forage for silkworm Bombyx mori L.. The mulberry leaves were the important material base of national sustainable development of practicing sericulture and silk industry. With the help of great progress in biotechnology, industry development of mulberry would be accelerated for using diversification strategies. The foliage is not only as the forage of silkworm Bombyx mori L., the value of mulberry in other industries has attracted more and more attentions by scientists.The protein content of the mulberry leaf (dry matter) is as high as24%-32%and the mulberry leaf with high phosphorus content has been usually used as feed, according to determination the phosphorus-containing of its tender leaf (fresh matter) is up to0.27%, and the phosphorus-containing of its mature leaf (fresh matter) is up to0.18%. However, the phosphorus of plant was mainly existed in phytic acid. Phytic acid is one of anti-nutritional factor; it had effects on the absorption of mineral elements by animals. The phosphorus from phytic acid could not be used by monogastric animal when the plant feedstuffs are provided as food or feed, and it caused the environment of phosphorus pollution. Meanwhile. the phosphorus element is the third position nutrients in animal feed after energy and protein:it is very important and composed of the tooth, bone and membrane of animal as well as nucleic acid. The phosphorus also participates in the important metabolism process s of growth and development. It is not only seriously affect the growth and development of animal but also leading to the lowest of performance when lack of phosphrus element. Thus how to use the phosphrus element of the plant feedstuffs effectively is a very important research topic.This study has developed around how to make use of phytase and then the systematic deep research has been done completely based on reliability. Meanwhile we had been obtained a good research achievement. The main results were briefly summarized as follws:1. Analysis of the Contents of Phytic acid in Mulberry Leaves by Iron Pectrophotometric MethodWe analyzed the different conditional factor on the yield of phytic acid from mulberry leaves by means of Box-Behnken model of the Design-Expert8.0.4Trial software, and the results showed that the order of the effective factors of phytic acid extraction was ratio of solute to material, hydrochloric acid percentage and extraction time, respectively. The optimum condition for the extraction was as following:1.25%hydrochloric acid and10%sodium sulfate, extraction time2.18h, ratio of solute to material0.05g/mL. Based on the optimum condition, we measured the phytic acid content in the leaves of five mulberry varieties and5instar-old spring silkworm excrement by colorimetric method. The average phytic acid content of five mulberry varieties are3.98mg·g-1in spring mature leaves,3.55mg·g-1in spring tender leaves,3.75mg·g-1in autumnal mature leaves and3.52mg·g-1in autumnal tender leaves, respectively. Meanwhile, the phytic acid content of silkworm excerment is3.78mg·g-1, considerably closed to that of mulberry leaves. The result showed that the phytic acid content was continuously increasing with the mulberry leaves in maturity, and the silkworm couldn't absorption and utilization the phosphorus phytic acid.2. Screened a Bacterial Strain from Soil and Cloned the phyC gene from the Genome of this StrainA bacterial strain designated WYCQ02, producing neutral phytase, was obtained from soil samples using strictly high-temperature screening. It was identified as Bacillus based on the16S rDNA and the physiological and biochemical characteristics. A1.2kb DNA fragment named phyC-WYCQ02was amplified from the genomic DNA of WYCQ02by PCR and the results from the sequence analysis of this fragment showed it contained an1152-bp long open reading frame (ORF). The ORF encoded a polypeptide of383amino acid residues with a putative signal peptide of26amino acids, the GenBank number is FJ986327and amino acid sequence number is ACR78677. The secondary structure of amino acid containing rich Random coil and Extended strand, just including a little amount of Alpha helix and Beta turn. The N-glycosylation site (Asn-Xaa-Ser/Thr) was forecast by NetNGlyc1.0, the result showed that it has four potential N-glycosylation sites; it located in95,112,217and255sites at the amino acid initial site respectively. It was beneficial to the folding and stability of protein.3. Expression of Neutral Phytase Gene from Bacillus sp. in Escherichia coli In order to obtain stability of phytase with highly active, the DNA fragments from phyC gene (FJ986327) from Bacillus sp. of the coding sequence (CDS) without its signal peptide sequence were amplified by polymerase chain reaction (PCR) and cloned into E. coli expression vector pET-28a (+), and then the recombinant vectors was transformed into the E. coli BL21(DE3) strain, and optimization of expression conditions for efficient expression. By0.5mmol/L IPTG induction at37℃for4h could obtain more protein but most of them belong to the inclusion body, and by0.5mmol/L IPTG induction at25℃for6h could obtain more soluble protein. The recombinant protein was purified by Ni-NTA affinity chromatography. Its optimum temperature for phytase activity was55℃, and more than20%of its original activity remained after incubation at70℃for10min, and there was no phytase activity when incubation for60min. The optimum pH for enzyme activity was6.0～7.0and more than80%of the enzyme activity was retained from5.5-9.0. The enzyme has some acid resistance, more than70%and40%of its original activity remained after incubation at pH5.0～10.0and pH2.0～4.0for60min. Our results provide useful information for further application of neutral phytase.4. Secretory Expression of Neutral Phytase Gene from Bacillus sp. in Pichia pastorisA pair of primers was designed to clone the phytase phyC fragment without signal peptide sequence according to the phyC gene complete sequences came from the Bacillus strain WYCQ02. Then the phyC fragment was cloned into pPIC9K expression vector of Pichia pastoris. The pPIC9K-phyC was linearized and transformed into Pichia pastoris GS115strain by electroporation. The phyC gene was integrated with chromosome of P.pastoris by PCR. Positive recombinant strains was screened and purified by culturing on MM plate and MD plate as well as YPD-phytic acid calcium salt plate. Next, the positive strain was cultured in BMGY culture medium and induced by methanol. The results showed that the protein was expressed secretively with phytase activity. The recombinant protein was purified by Ni-NTA affinity chromatography. Its optimum temperature for phytase activity was55℃. The enzyme has some acid resistance and with higher activity in the pH7-8. This phyC gene had been certificated as the National Invention Patent of China (Patent Application number:200910103029.1), and it had obtained the patent of invention in China in December8,2010, Publicity number:ZL200910103029.1, Certificate number:710888.5. Studies on phyC Gene Transformation in MulberryIn order to confirm the optimum selective concentration of antibiotic in genetic transformation, kanamycin was selected as a case in this paper, and meanwhile the antibiotic concentration did not cause any injury in explants. It had been found that the optimum selective concentration of kanamycin was50mg/L. It is reported that mulberry was sensitivity in Agrobacterium immersing experiments, and using the rapid and efficient plant regeneration system which established in our laboratory as the receptor of transformation. The phyC gene genetic transformation in mulberry Morus L. was studied by agrobacterium-mediated in order to improve the utilization ratio of mulberry leaves'phosphrus, and at last we obtained15plantlets of phyC gene transformation which have PCR positive. The results show that it is feasible to gene transformat into mulberry by Agrobacterium-mediated Transformation.In this study we used the pollen-mediated reference to the method of Li Jun and Zhao Ai-chun et al. to do the research of gene transformation. Introduction of phyC Gene from Bacillus sp. into the hybrid mulberry seed of Red No.1and Breeding No.2by pollen-mediated. A total number of1301hybrid seeds were obtained and some seed had been selected by kanamicy.11plantlets were obtained under PCR analysis. Thus it is verified the mulberry of gene transformation in our laboratory. The data laid the foundations for further study of the mulberry of gene transformation in our laboratory.