中性植酸酶基因克隆表达、功能分析及对桑树遗传转化的研究
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摘要
栽桑养蚕最早始于我国,是中华民族对人类文明的伟大贡献之一。桑树(Morus L.)属多年生双子叶木本植物,是一种重要的木本经济作物,其收获物——桑叶是家蚕的绝好饲料,是我国蚕桑丝绸产业可持续发展的重要物质基础。随着科技的发展,桑叶不仅是作为家蚕的饲料,而且已用作畜禽饲料,使桑树产业向着多元化方向发展,桑树的多用途价值已越来越受到广大科学者的关注。
桑叶不仅蛋白质含量高(干物高达24%-32%),而且也是一种含磷量较高的饲料用叶,其嫩叶(鲜物)的含磷量高达0.27%,成熟叶(鲜物)的含磷量高达0.18%。然而植物中的磷有相当部分是以植酸的形式存在,植酸是一种抗营养因子,能够抑制动物体对矿质元素的吸收。当植物性饲料被单胃动物采食后,其有机磷不能被吸收利用,而要向外排泄,污染环境水质,同时磷在动物饲料中是仅次于能量和蛋白质的第三位营养素,占有举足轻重的地位,它参与动物牙齿、骨骼及细胞膜的构成,并是遗传物质核酸的组成成分,参与重要的生长发育代谢过程。磷元素缺乏将严重影响动物生长发育,导致动物的生产性能低下,因此,如何最大程度的利用植食性饲料中所含的磷元素,是极为重要的研究课题。
本研究围绕如何利用桑叶植酸磷这一课题,开展了较为深入系统的研究,并取得了较好的研究成绩,现将其研究要点总结如下：
1.利用测铁分光光度法测定桑叶中植酸的含量
本研究运用Design-Expert8.0.4Trial软件的Box-Behnken模型,分析不同提取条件因子对桑叶植酸提取效果的影响作用,为料液质量浓度>提取液中盐酸的浓度>浸提时间,最佳提取条件是：提取液为1.25%盐酸+10%硫酸钠,浸提时间为2.18h,料液质量浓度为0.05g/mL。采用此优化条件提取桑叶和蚕粪中的植酸,并应用比色法测定5个桑品种和5龄幼虫蚕粪中的植酸含量,5个桑品种春季成熟叶、嫩叶以及秋季成熟叶、嫩叶中的植酸平均质量比分别为3.98、3.55、3.75、3.52mg·g-1；蚕粪中植酸的含量为3.78mg·g-1,与桑叶中的植酸含量基本接近。检测结果说明桑叶中的植酸随着桑叶的成熟其含量不断增加,实验结果表明家蚕对桑叶中植酸态磷几乎不能利用。
2.枯草芽孢杆菌的筛选及植酸酶phyC基因的克隆
本研究以来自于西南大学后山池塘边枯草丛的土壤为材料,通过两次高温加热筛选,从中获得一株命名为WYCQ02的细菌,利用改良的schaeffer-fulton染色法对该细菌进行染色发现菌体呈红色杆状且较长,芽孢呈蓝色的椭圆形；镜检发现菌体周身长有鞭毛,具运动性,穿刺接种发现该菌为好氧性细菌,结合16S rDNA序列分析在NCBI的比对结果我们将其鉴定为枯草芽孢杆属的一种。
利用NCBI中已报道的中性植酸酶基因序列设计引物,采用PCR方法,从WYCQ02菌株的基因组中克隆获得一植酸酶基因,并向NCBI提交登录,基因序列号为FJ986327,氨基酸序列号为ACR78677。该基因序列包含一完整的1152bp长的开放阅读框,编码357个氨基酸的成熟肽和26个氨基酸的信号肽。与芽孢杆菌属植酸酶的同源性较高,其二级结构含有丰富的随机卷曲和衍生链,仅有少量的α-螺旋和β-转角。利用NetNGlyc1.0在线分析软件对其N-端糖基化位点(Asn-Xaa-Ser/Thr)进行预测发现存在4个潜在的N-端糖基化位点,即分别位于氨基酸起始位点的第95位点,112位点,217位点和255位点的Asn残基均可以被糖基化,这将有利于蛋白的折叠和稳定性。
3.芽孢杆菌植酸酶phyC基因的原核表达
本研究根据前述基因序列的结构以及原核表达载体pET-28a(+)的多克隆位点设计引物,采用PCR方法扩增芽孢杆菌中性植酸酶phyC基因的成熟肽编码序列,然后将其克隆进原核表达载体pET-28a(+),并转化大肠杆菌BL21(DE3)进行表达。在37℃条件下以0.5mmol/L IPTG诱导4h能够获得大量包涵体蛋白,在25℃条件下以0.5mmol/L IPTG诱导6h有利于可溶性蛋白的获得。利用Ni-NTA亲和纯化重组植酸酶产物,对纯化的中性植酸酶的部分酶学性质进行分析表明：该酶的最适反应温度为55℃,并具有一定的耐热性,在70℃处理10min可保持20%以上的酶活性；最适pH值在6～7之间,当pH值为5.5～9.0能保持80%以上的酶活性；该酶还具有一定的耐酸、碱能力,在pH5.0～10.0之间处理60min该酶仍能保持70%以上的活力,在pH2.0～4.0之间能保持40%以上的活力。
4.芽孢杆菌植酸酶phyC基因的真核表达
本研究根据芽孢杆菌植酸酶phyC基因全序列和pPIC9K的多克隆位点设计引物,采用PCR方法从pGEM-phyC质粒分别获得含信号肽的phyC基因开放阅读框和成熟肽编码序列,然后将其连接到毕赤酵母表达载体pPIC9K的多克隆位点上,利用限制性核酸内切酶Sac I将表达载体pPIC9K-phyC线性化后,经Bio-Rad MicroPulserTM电转仪电击转化入巴斯德毕赤酵母GS115。经菌落PCR和MD/MM平板筛选获得了阳性酵母表达菌株,利用含G418的平板筛选出phyC基因多拷贝酵母菌株后,再利用YPD-植酸钙平板筛选获得具胞外表达植酸酶活性且较高的酵母菌株,然后在BMGY和BMMY培养基中利用甲醇诱导获得了有生物学活性的芽孢杆菌植酸酶的分泌表达。表达的蛋白经Ni-NTA亲和柱纯化后,对部分酶学性质做了分析,发现其酶促反应的最适温度为55℃,pH值在7-8之间均有相当高的酶活性,并且该酶亦具有一定的耐酸性和耐碱性。通过对原核和真核表达的对比发现,真核表达的植酸酶蛋白仅在热稳定性上有所提高。由此将我们获得的phyC基因,向中华人民共和国国家知识产权局进行了发明专利申请登记,专利申请登记号：200910103029.1,并于2010年12月8日获得国家发明专利授权,专利号：ZL200910103029.1,证书号第710888号。
5.芽孢杆菌植酸酶phyC基因转化桑树的初步研究
在以抗生素为筛选标记的基因转化中,确定适宜的筛选压力是提高基因转化率的前提,并且还不能对外植体有所伤害。本研究通过实验确定了卡那霉素筛选浓度为50mg/L,经过前人的不少研究表明桑树是一种易感农杆菌的植物。利用本研究室多年研究优化的桑组培再生体系获得桑树外植体丛生芽,以此作为遗传转化的受体,构建pCAMBIA-2300-phyC载体,利用农杆菌介导的基因转化法,共转化丛生芽527个,得到45株phyC遗传基因转化组培桑苗。经鉴定检测,其中PCR阳性的phyC遗传基因转化植株为15株,其PCR阳性率为2.85%。实验结果表明,农杆菌介导基因转化桑树是可行的。
本研究借鉴本研究室李军、赵爱春等的花粉介导法将phyC基因导入红果1号和育2号杂交桑种子中,共收获1301粒种子,其中500粒种子在萌发期用卡那霉素筛选,获得64株桑苗移栽大田；另外801粒桑种,直接播种在大田中进行精心管理。最终总共获得154株遗传转化实生苗,于2011年8月至11月连续逐株进行PCR检测,共获得了11株具有PCR阳性的phyC基因转化桑苗植株,花粉介导phyC基因的PCR阳性植株与杂交桑种粒数的比例为0.85%。下一步的工作还需要对这些PCR阳性植株进行各种分子杂交技术检测,并进行酶活性的测定验证,最终获得具有植酸酶活性的遗传转化植株。
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 Method
We 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 Strain
A 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 pastoris
A 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 Mulberry
In 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.
桑叶不仅蛋白质含量高(干物高达24%-32%),而且也是一种含磷量较高的饲料用叶,其嫩叶(鲜物)的含磷量高达0.27%,成熟叶(鲜物)的含磷量高达0.18%。然而植物中的磷有相当部分是以植酸的形式存在,植酸是一种抗营养因子,能够抑制动物体对矿质元素的吸收。当植物性饲料被单胃动物采食后,其有机磷不能被吸收利用,而要向外排泄,污染环境水质,同时磷在动物饲料中是仅次于能量和蛋白质的第三位营养素,占有举足轻重的地位,它参与动物牙齿、骨骼及细胞膜的构成,并是遗传物质核酸的组成成分,参与重要的生长发育代谢过程。磷元素缺乏将严重影响动物生长发育,导致动物的生产性能低下,因此,如何最大程度的利用植食性饲料中所含的磷元素,是极为重要的研究课题。
本研究围绕如何利用桑叶植酸磷这一课题,开展了较为深入系统的研究,并取得了较好的研究成绩,现将其研究要点总结如下：
1.利用测铁分光光度法测定桑叶中植酸的含量
本研究运用Design-Expert8.0.4Trial软件的Box-Behnken模型,分析不同提取条件因子对桑叶植酸提取效果的影响作用,为料液质量浓度>提取液中盐酸的浓度>浸提时间,最佳提取条件是：提取液为1.25%盐酸+10%硫酸钠,浸提时间为2.18h,料液质量浓度为0.05g/mL。采用此优化条件提取桑叶和蚕粪中的植酸,并应用比色法测定5个桑品种和5龄幼虫蚕粪中的植酸含量,5个桑品种春季成熟叶、嫩叶以及秋季成熟叶、嫩叶中的植酸平均质量比分别为3.98、3.55、3.75、3.52mg·g-1；蚕粪中植酸的含量为3.78mg·g-1,与桑叶中的植酸含量基本接近。检测结果说明桑叶中的植酸随着桑叶的成熟其含量不断增加,实验结果表明家蚕对桑叶中植酸态磷几乎不能利用。
2.枯草芽孢杆菌的筛选及植酸酶phyC基因的克隆
本研究以来自于西南大学后山池塘边枯草丛的土壤为材料,通过两次高温加热筛选,从中获得一株命名为WYCQ02的细菌,利用改良的schaeffer-fulton染色法对该细菌进行染色发现菌体呈红色杆状且较长,芽孢呈蓝色的椭圆形；镜检发现菌体周身长有鞭毛,具运动性,穿刺接种发现该菌为好氧性细菌,结合16S rDNA序列分析在NCBI的比对结果我们将其鉴定为枯草芽孢杆属的一种。
利用NCBI中已报道的中性植酸酶基因序列设计引物,采用PCR方法,从WYCQ02菌株的基因组中克隆获得一植酸酶基因,并向NCBI提交登录,基因序列号为FJ986327,氨基酸序列号为ACR78677。该基因序列包含一完整的1152bp长的开放阅读框,编码357个氨基酸的成熟肽和26个氨基酸的信号肽。与芽孢杆菌属植酸酶的同源性较高,其二级结构含有丰富的随机卷曲和衍生链,仅有少量的α-螺旋和β-转角。利用NetNGlyc1.0在线分析软件对其N-端糖基化位点(Asn-Xaa-Ser/Thr)进行预测发现存在4个潜在的N-端糖基化位点,即分别位于氨基酸起始位点的第95位点,112位点,217位点和255位点的Asn残基均可以被糖基化,这将有利于蛋白的折叠和稳定性。
3.芽孢杆菌植酸酶phyC基因的原核表达
本研究根据前述基因序列的结构以及原核表达载体pET-28a(+)的多克隆位点设计引物,采用PCR方法扩增芽孢杆菌中性植酸酶phyC基因的成熟肽编码序列,然后将其克隆进原核表达载体pET-28a(+),并转化大肠杆菌BL21(DE3)进行表达。在37℃条件下以0.5mmol/L IPTG诱导4h能够获得大量包涵体蛋白,在25℃条件下以0.5mmol/L IPTG诱导6h有利于可溶性蛋白的获得。利用Ni-NTA亲和纯化重组植酸酶产物,对纯化的中性植酸酶的部分酶学性质进行分析表明：该酶的最适反应温度为55℃,并具有一定的耐热性,在70℃处理10min可保持20%以上的酶活性；最适pH值在6～7之间,当pH值为5.5～9.0能保持80%以上的酶活性；该酶还具有一定的耐酸、碱能力,在pH5.0～10.0之间处理60min该酶仍能保持70%以上的活力,在pH2.0～4.0之间能保持40%以上的活力。
4.芽孢杆菌植酸酶phyC基因的真核表达
本研究根据芽孢杆菌植酸酶phyC基因全序列和pPIC9K的多克隆位点设计引物,采用PCR方法从pGEM-phyC质粒分别获得含信号肽的phyC基因开放阅读框和成熟肽编码序列,然后将其连接到毕赤酵母表达载体pPIC9K的多克隆位点上,利用限制性核酸内切酶Sac I将表达载体pPIC9K-phyC线性化后,经Bio-Rad MicroPulserTM电转仪电击转化入巴斯德毕赤酵母GS115。经菌落PCR和MD/MM平板筛选获得了阳性酵母表达菌株,利用含G418的平板筛选出phyC基因多拷贝酵母菌株后,再利用YPD-植酸钙平板筛选获得具胞外表达植酸酶活性且较高的酵母菌株,然后在BMGY和BMMY培养基中利用甲醇诱导获得了有生物学活性的芽孢杆菌植酸酶的分泌表达。表达的蛋白经Ni-NTA亲和柱纯化后,对部分酶学性质做了分析,发现其酶促反应的最适温度为55℃,pH值在7-8之间均有相当高的酶活性,并且该酶亦具有一定的耐酸性和耐碱性。通过对原核和真核表达的对比发现,真核表达的植酸酶蛋白仅在热稳定性上有所提高。由此将我们获得的phyC基因,向中华人民共和国国家知识产权局进行了发明专利申请登记,专利申请登记号：200910103029.1,并于2010年12月8日获得国家发明专利授权,专利号：ZL200910103029.1,证书号第710888号。
5.芽孢杆菌植酸酶phyC基因转化桑树的初步研究
在以抗生素为筛选标记的基因转化中,确定适宜的筛选压力是提高基因转化率的前提,并且还不能对外植体有所伤害。本研究通过实验确定了卡那霉素筛选浓度为50mg/L,经过前人的不少研究表明桑树是一种易感农杆菌的植物。利用本研究室多年研究优化的桑组培再生体系获得桑树外植体丛生芽,以此作为遗传转化的受体,构建pCAMBIA-2300-phyC载体,利用农杆菌介导的基因转化法,共转化丛生芽527个,得到45株phyC遗传基因转化组培桑苗。经鉴定检测,其中PCR阳性的phyC遗传基因转化植株为15株,其PCR阳性率为2.85%。实验结果表明,农杆菌介导基因转化桑树是可行的。
本研究借鉴本研究室李军、赵爱春等的花粉介导法将phyC基因导入红果1号和育2号杂交桑种子中,共收获1301粒种子,其中500粒种子在萌发期用卡那霉素筛选,获得64株桑苗移栽大田；另外801粒桑种,直接播种在大田中进行精心管理。最终总共获得154株遗传转化实生苗,于2011年8月至11月连续逐株进行PCR检测,共获得了11株具有PCR阳性的phyC基因转化桑苗植株,花粉介导phyC基因的PCR阳性植株与杂交桑种粒数的比例为0.85%。下一步的工作还需要对这些PCR阳性植株进行各种分子杂交技术检测,并进行酶活性的测定验证,最终获得具有植酸酶活性的遗传转化植株。
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 Method
We 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 Strain
A 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 pastoris
A 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 Mulberry
In 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.
引文
[1]何文寿.植物营养学通论[M].银川：宁夏人民出版社,2004,148-155.
[2]吴平,印莉萍,张立平等.植物营养分子生理学[M].北京：科学出版社,2001,102-103.
[3]呙于明,汤芹.植酸酶在养鸡业的应用研究(综述)[J].中国畜牧杂志,1997,33(6)：48-50.
[4]Purva Vats, Bharat Bhushan, U.C. Banerjee. Studies on the dephosphorylation of phytic acid in livestock feed using phytase from Aspergillus niger van Teighem [J]. Bioresource Technology,2009,100 (1):287-291.
[5]王红宁,黄勇,陈惠等.饲用微生物植酸酶的研究进展(综述)[J].国外畜牧学-饲料,1999,(3)：9-10.
[6]王红宁,吴琦等.黑曲霉N25植酸酶phvA基因的克隆及序列分析[J].微生物学报,2001,41(3)：310-314.
[7]Bergman E L, Autio K, Sanberg A S. Optimal conditions for paytate degradation, estimation of phytase activity, and localization of phytate in barley (cv. Blenheim) [J]. J Agric Food Chem,2000,48(10):4647-4655.
[8]赵玉生.植酸在食品工业中的应用[J].西部粮油科技,2000,25(6)：51-52.
[9]傅启高,李慧荃.三氯化铁比色法测植酸含量的研究[J].营养学报,1997,19(2)：210-220.
[10]刘巧茹,石起增,董文举.硝酸钍测定植酸含量方法的校正[J].平顶山学院学报,2006,21(5)：38-40.
[11]Dost K, Tokul O. Determination of phytic acid in wheat and wheat products by reverse phase high performance liquid chromatography [J]. Anal. Chim. Acta,2006,558 (1-2):22-27.
[12]肖传豪,陈来成.快速简便测定植酸的方法[J].化工时刊,2010,24(8)：66--68.
[13]Mason T J, Paniwnyk L, Lorimer J P. The uses of ultrasound in food technology [J]. Ultrasonic Chemistry, 1996,3(3):253-260.
[14]武雪芬,李玉贤,李桂兰.利用植酸对磺基水杨酸铁的脱色作用比色法测定植酸含量[J].河南科学,1996,14(1)：50-52.
[15]郑穗平.饲料工业酶技术[M].化学工业出版社,2008,8：29-30.
[16]吴谋成,袁俊华.植酸的毒理学评价和食用安全性[J].食品科学,1997,18(2)：46-49.
[17]Pallauf J, Rimbach G. Nutritional significance of phytic acid and Phytase [J]. Arch Tieremahr,1997,50(4): 301-319.
[18]黄君霆,朱万民,向仲怀等.中国蚕丝大全[M].四川科技出版社,1996,278-294.
[19]Boling S D, Douglas M W, Johnson M L, et al. The effects of dietary available phosphorus levels and phytase performance of young and older laying hens [J]. Poult Sci,2000,79:224-230.
[20]Cao L., Wang W., Yang C., et al. Application of microbial phytase in fish feed [J]. Enzyme and Microbial Technology,2007,40:497-507.
[21]Mallin M A. Impacts of industrial animal production on rivers and estuaries [J]. Am Sci,2000,88:26-37.
[22]Mullaney E. J, Daly C, et al. Advances in phytase esearch [J]. Adv Appl Microbiol,2000,47:157-199.
[23]Ullah A H, Sethumadhavan K. PhyA gene product of Aspergillus ficuum and Peniophora lycii produces dissimilar phytases [J]. Biochem. Biophys. Res. Commun.2003.303(2):463-468.
[24]Simons P C, Versteegh H A, Jongbloed A W. et al. Improvement of phosphorus availability by microbial phytase in broilers and pigs [J]. British Journal of Nutrition.1990.64(2):525-540.
[25]Reddy N R, Sathe S K, Salunkhe D K. Phytates in cereals and legumes [J]. Adv Food Res,1982,28:1-92.
[26]Kim Y O, Kim H K, Yu J H, et al. Cloning of the thermostable phytase gene (phy) from Bacillus sp.DS11 and its overexpression in Escherichia coli [J]. FEMS Microbiology Letters,1998,162:185-191.
[27]Ravindran V, Kornegay E T, Denbow D M, et al. Response of turkey poults to tiered levels of Natuphos phytase added to soybean mealbased semi-purified diets containing three levels of nonphytate phosphorus [J]. Poult Sci,1995,74:1843-1854.
[28]农业部办公厅：关于印发《饲用植酸酶使用指南》的通知,农办牧[2008]29号,中华人民共和国农业部公报,2008,6：37-38；
[29]Cromwell G L, Coffey R D, Parker G R, et al. Effiency of phytase in improving the bioavailability of phosphorus in soybean meal and corn-soybean meal diets for pigs [J]. J Anim Sci,1993,71:1831-1840.
[30]Vucenik L, Shamsuddin A M. Cancer inhibition by inositol Hexaphosphate (IP6) and inositol:From Laboratory to Clinic [J]. J. Nutr.,2003,133:3778-3784.
[31]Hieh T R, Ware J H. Survey of microorganisms for the production of extracellular phytase [J]. Appl. Microbiol., 1968,16:1348-1351.
[32]周庆安,姚军虎,何瑞国.植物性植酸酶及其应用[J].广东饲料,2004,4(14)：24-25.
[33]周庆安等.植物植酸酶减少养殖业磷排泄的研究[J].中国青年农业科学学术年报,2004,149-152；
[34]冯定远.酶制剂在饲料工业中的应用[M].北京：中国农业科学技术出版社,2005,11：299-309.
[35]Iqbal T H, Lewis K O. phytase activity in the human and rat small intestine [J]. Gut,1994,35(9):1233-1236.
[36]Ghareib M. Biosynthesis, Purification and Some Properties of Eatracellular Phytase from Aspergillus carneus [J]. Acta Microbiol. Hung.,1990,37(2):159-164.
[37]Wyss M, Pasamontes L, Remy R, et al. Comparison of the thermostability properties of three acid phosphatases from molds:Aspergillus fumigatus phytase, A. niger phytase and A. niger pH 2.5 acid phosphatase [J]. Appl. Environ. Microbiol.,1998,64:4446-4451.
[38]Han Y M, Roneker K R, et al. Adding wheat middling, microbial phytase, and citric acid to conr-soybean meal diets for growing pigs may replace inorganic phosphorus supplementation [J]. J. Anim. Sci.,1998,76: 2649-2656.
[39]Nakashima B A, Mcallister T A, Shanna R, et al. Diversity of phytase in the rumen [J]. Microb Ecol.,2007, 53(1):82-88.
[40]Schenk G, Guddat LW, Ge Y, et al. Identification of mammalian-like purple acid phoshpatases in a wide range of plants [J]. Gene,2000,250:117-125.
[41]Fu S, Sun J, Qian L, et al. Bacillus phytase:present scenario and future perspectives [J]. Appl Biochem Biotechnol,2008,151:1-8.
[42]Gylati H K, Chadha B S, Saini H S. Production and characterization of thermostable alkaline phytase from Bacillus laevolacticus isolated from rhizosphere soil [J]. J Ind Microbiol Biotechnol.,2007,34:91-98.
[43]Scott J J. A calcium-activated from pollen of Lilium longiflorum [J]. Plant Physiol,1986,82(1):333-335.
[44]Huang H, Shao N, Wang Y, et al. A novel beta-propeller phytase from Pedobacter nyacknsis MJ11 CGMCC 2503 with potential as an aquatic feed additive [J]. Appl Microbiol Biotechnol.2009.83:249-259.
[45]Lim B L, Yeung P, Cheng C, et al. Distribution and diversity of phytate-mineralizing bacteria [J]. ISME J.2007. 1:321-330.
[46]Choi Y M, Suh H J, Kim J M. Purification and properties of extracellular phytase from Bacillus sp. KHU-10 [J]. J Protein Chem,2001,20:287-292.
[47]Powar V K, Jagannathan V. Purification and properties of phytate-specific phosphatase from Bacillus subtilis [J]. J Bacteriol.1982,151:1102-1108.
[48]王亚茹,姚斌等.枯草芽孢杆菌中性植酸酶的纯化和酶学性质[J].微生物学报,2001,41(2)：198-203.
[49]Tye A J, Siu F K, Leung T Y, et al. Molecular cloning and the biochemical characterization of two novel phytases from B. subtilis 168 and B. licheniformis [J]. Appl Microbiol Biotechnol,2002,59:190-197.
[50]Ha N C, Oh B C, Shin S, et al. Crystal structures of a novel, themostable phytase in partially and fully calcium-loaded states [J]. Nature Struct Biol,2000(7):147-184.
[51]Kerovuo J, Lappalainen I, Reinikainen T. The metal dependence of Bacillus subtilis phytase [J]. Biochem Biophys Res Commun,2000,268:365-369.
[52]Shin S, Ha N C, Oh B C, et al. Enzyme mechanism and catalytic property of beta propeller phytase [J]. Structure,2001,9(9):851-858.
[53]Oh B C, Kim M H, Yun B S, et al. Ca(2+)-inositol phosphate chelation mediates the substrate specificity of beta-propeller phytase [J]. Biochemistry,2006,45(31):9531-9539.
[54]Greiner R, Farouk A, Alminger M L, et al. The pathway of dephosphorylation of myo-insitol hexakisphosphate by phytatedegeading enzymes of different Bacillus spp [J]. Can J Microbiol.,2002,48:986-994.
[55]彭景.烹饪营养学[M].中国纺织出版社,2008,68-69.
[56]林志勋,江世楷.禽畜消化道、肠道细菌和其消化能力之关系[J].中国畜牧杂志,1996,28(6)：17-27.
[57]姚斌,杨培龙,黄火清.中性植酸酶的研究与前景[J].生物产业技术,2010,4：42-47.
[58]伍喜林,陈贵才.应用动物营养学[M].四川科学技术出版社,2005,9：217-218.
[59]Lei X G, Porres J M, Mullaney E J, et al. Source, Structure and Application. Chapter 29, Industrial Enzyme Structure, Function and Application:Edited by Polaina J, MacCabe A.P.2007.
[60]Coello P, Maughan J P, Mendoza A, et al. Generation of low phytic acid Arabidopsis seeds expressing an E.coli phytase during embryo development [J]. Seed Science Research,2001,11 (1):185-291.
[61]Streb H, Irvine R F, Berridge M J, et al. Release of Ca2+ from a nonmitochondrial intracellular store in pancreatic acinar cells by inositol-1,4,5-trisphosphate [J]. Nature,1983,306:67-69.
[62]Claxon A, Morris C, Blake D, et al. The anti-inflammatorv effects of D-myo-inositol-1.2.6-trisphosphate (PP56) on animal models of inflammation [J]. Agents Actions.,1990,29:68-70.
[63]杨平平,许正宏,王燕等.微生物植酸酶进展[J].微生物学通报,2004,31(3)：111-115.
[64]王红艳,王云飞,王申涛.植酸酶的研究进展及应用[J].中国酿造,2010,4：24-25.
[65]pET System Manual.2003,3-5.
[66]Cregg J M. Vedvick T S, Raschke W C. Recent Advances in the expression of foreign genes in Pichia pastoris [J]. Bio/Technology.1993.11(8):905-910.
[67]Peng Y, Bu W, Kang L Y. Methylotrophic yeast system [J]. Biotechnol Information,2000,1:38-41.
[68]Zhuang S M, Landegent J E. Verschuerren C A. et al. Apoptin, a protein encoded by chicken anemia virus, induces cell death in human hematologic malignant cells [J]. Leukemia,1995,1:118-120.
[69]胡世界,罗素兰,张吉贞等.巴斯德毕赤酵母表达系统及其高水平表达策略[J].生物技术,2007,17(6)：78-83.
[70]Patricia S. Vary, Rebekka Biedendieek, Tobias Fuerch, et al. Bacillus megaterium from simple soil bacterium to industrial protein production host [J]. Applied Microbiology and Biotechnology,2007,76(5):957-967.
[71]Rygus T, Hillen W. Inducible high-level expression of heterologous genes in Bacillus megaterium using the regulatory elements of the xylose-utilization operon [J]. Applied Microbiology and Biotechnology,1991,35, 594-599.
[72]Wittchen K D, Meinhardt F. Inactivation of the major extracellular protease from Bacillus megaterium DSM319 by gene replacement [J]. Applied Microbiology and Biotechnology,1995,42(6):871-877.
[73]姚斌,袁铁铮,王元火等.来源于Bacillus的中性植酸酶基因的克隆及在大肠杆菌中的表达[J].生物工程学报,2001,17(1)：12-15.
[74]吴琦,王红宁,刘世贵等.枯草芽孢杆菌植酸酶基因在大肠杆菌中的表达及对酶热稳定性的影响[J].高技术通讯,2004,14(5)：23-27.
[75]邹立扣,王红宁,吴琦等.植酸酶phyC基因表达载体的构建及其在E.coli中的表达[J].生物技术,2004,14(3)：11-14,
[76]牟琳.枯草芽孢杆菌WHNB02中性植酸酶基因在巨大芽孢杆菌中的表达研究.硕士学位论文.四川.四川农业大学.2008.
[77]Guerrero-Olazaran M, Rodriguez-Blanco L, Carreon-Trevino J G, et al. Expression of a Bacillus Phytase C Gene in Pichia pastoris and Properties of the Recombinant Enzyme [J]. Appl. Environ. Microbiol.,2010, 76(16):5601-5608.
[78]Zou L K, Wang H L, Pan X, et al. Design and Expression of a Synthetic phyC Gene Encoding the Neutral Phytase in Pichia pastoris [J]. Acta Biochimica et Biophysica Sinica,2006,38(11):803-811.
[79]Larrick J W, Thomas D W. Producing proteins in transgenic plants and animals [J]. Curr. Opin. Biotechnol., 2001,12:411-418.
[80]Pen J, Verwoerd T C, van Paridon P A, et al. Phytase-containing transgenic seeds as anovel feed additive for improved phosphorus utilization [J]. Bio/Technology.,1993,11:811-814.
[81]Verwoerd T C, Van Paridon P A, Van Ooyen A J J, et al. Stable accumulation of Aspergillus niger phytase in transgenic tobacco leaves [J]. Plant Physiol.1995,109:1199-1205.
[82]Ullah A H J, Sethumadhavan K, Mullaney E J, et al. Characterization of recombinant fungal phytase (phyA) expressed in tobacco leaves [J]. Biochem Biophys Res Commun.,1999,264:201-206.
[83]Mudge S R, Smith F W, Richardson A E. Root-specific and phosphate-regulated expression of phytase under the control of a phosphate tr ansporter promoter enables Arabidopsis to grow on phytate as a sole P source [J]. Plant Science,2003,165:871-878.
[84]Gutknecht K. Green genes:alfalfa biofanning is about to take root [J]. Wise Agrict.,1997,3:8-10.
[85]Brinch-Pedersen H, Olesen A, Rasmussen S K, et al. Generation of transgenic wheat (Triticum aestivum L.) for constitutive accumulation of an Aspergillus phytase [J]. Mol Breed..2000.6:195-206.
[86]Ponstein A S, Bade J B, Verwoerd T C, et al. Stable expression of Phytase (phyA) in canola (Brassica napus) seeds:towards a commercial product [J]. Mol Breed.,2002,10:31-44.
[87]Denbow D M, Grabau E A, Lacy G H, et al. Soybeans transformed with a fungal phytase gene improve phosphorus availability for broilers [J]. Poultry Sci.,1998,77:878-881.
[88]Zhang Z B, Kornegay E T, Radcliffe J S, et al. Comparison of genetically engineered microbial and plant phytase for young broilers [J]. Poultry Sci.,2000,79:709-717.
[89]Hong C, Cheng K, Tseng T, et al. Production of two highly active bacterial phytases with broad pH optima in germinated transgenic rice seeds [J]. Transgenic Res.,2004,13:29-39.
[90]Xiao K, Harrison M, Wang Z Y. Transgenic expression of a novel M.truncatula phytase gene results in improved acquisition of organic phosphorus by Arabidopsis [J]. Planta.,2005,222:27-36.
[91]Xiao K, Zhang J H, Harrison M, et al. Ectopic expression of a phytase gene from Medicago truncatula Barrel Medic enhances phosphorus absorption in plants [J]. Journal Integrative Plant Biology.,2006,48(1):35-43.
[92]Li J, Hegeman C E, Hanlon R W, et al. Secretion of active recombinant phytase from soybean cell suspention cultures [J]. Plant Physiol.,1997,114:1103-1111.
[93]Austin S, Bingham E T, et al. Production and field performance of transgenic alfalfa(Medicago sativa L.) expressing alpha-amylase and manganese-dependent lignin peroxidase [J]. Euphytica.,1995,85:381-393.
[94]Ponstein A S, Bade J B, et al. Stable expression of Phytase (phyA) in canola (Brassica napus) seeds:towards a commercial product [J]. Molecular Breeding.,2002,10:31-44.
[95]Chen R M, Yao B, Fan Y L, et al. Transgenic maize plants expressing a fungal phytase gene [J]. Transgenic Res.,2008,17:633-643.
[96]范云六：利用玉米种子生物反应器成功培育出生产高活性植酸酶并稳定遗传的转基因玉米,新华网北京2007年9月10日电,记者姚润丰.
[97]谈建中,楼程富,钟名其等.大豆球蛋白基因表达载体的构建及对桑树的遗传转化[J].蚕业科学,1999,25(1)：6-11.
[98]陆小平,楼成富,王波等.用植株转化法将GUS基因导入桑树幼苗的研究[J].蚕业科学,2004,30(2)：120-132.
[99]Shalini L, Vibha G, Paramjit K. Overexpression of HVA1 gene from barley generates tolerance to salinity and water stress in transgenic mulberry (Morus indica) [J]. Transgenic Res.,2008,17:651-663.
[100]侯小峰.植酸酶之困[J].中国畜牧杂志,2008,44(8)：1-5.
[101]Greiner R, Carlson NG, Alminger M L. Stereospecificity of myo-inositol hexa phosphate dephosphorylation by phytate degrading enzyme by Escherichia coli [J]. J Biotech,2000,84:53-62.
[102]Purva V, Uttam C Banerje. Production studies and catalytic properties of phytases (myo-inositolhexakisphos-phate phosphohydrolases):an overview. Enzyme and Microbial Technology,2004,35(1):3-14.
[103]Afnah S, Yazid A M, Anis Shobirin M H, et al. Phytase:application in food industry. International Food Research Journal,2010,17:13-21.
[104]柯益富.桑树栽培及育种学[M].北京：中国农业出版社,1997：156-159.
[105]Maenz D D. Classen H L. Phytase activity in the small intestinal brush-border membrane of the chicken [J]. Poult Sci.1998,77:557-563.
[106]Naqvi S W A. Jayakumar D A, Narvekar P V, et al. Increased marine production of N2O due to intensifying anoxia on the Indian continental shelf [J]. Nature,2000,408:346-349.
[107]陆欣主编.十壤肥料学[M].北京：中国农业大学出版社,2002,208-227.
[108]Saad N. Esa N M. Ithnin H, et al. Optimization of optimum condition for phytic acid extraction from rice bran [J]. African Journal of Plant Science,2011,5(3):168-176.
[109]Kwanyuen P, Burton J W. A simple and rapid procedure for phytate determination in soybeans and soy products [J]. J Am Oil Chem Soc,2005,82(2):81-85.
[110]余安,王承明.超声辅助提取花生粕中植酸工艺优化[J].食品科学,2010,31(10)：179-183.
[111]沈维治,廖森泰,刘吉平等.用二次回归正交旋转组合设计优化桑叶多酚的提取工艺[J].蚕业科学,2009,35(3)：594-598.
[112]Camire A L, Clydesdale F M. Analysis of phytic acid in foods by HPLC [J]. J Food Sci,1982,47(2): 575-578.
[113]王忠.植物生理学[M].北京：中国农业出版社,2000：84-85.
[114]李朝霞,王爱民,李小敏.中性植酸酶高产菌株的筛选及产酶条件研究[J].微生物学通报,2007,34(4)：633-641.
[115]霍春雁,李锋,单冉东.生物化学实验方法和技术[M].北京：科学出版社.2002.
[116]吴琦.枯草芽孢杆菌植酸酶phyC基因的克隆及其表达研究[D].成都：四川大学,2004.
[117]陈艳,孙建义,赵学新等Bacillus amyloliquefaciens中性植酸酶基因的原核表达及蛋白纯化和性质[J].食品与生物技术学报,2005,24(2)：60-65.
[118]Chen X H, et al. Comparative analysis of the complete genome sequence of the plant growth-promoting bacterium Bacillus amyloliquefaciens FZB42 [J]. Nat Biotechnol,2007,25(9):1007-1014.
[119]Rao D E, Rao K. V, Reddy V D. Cloning and expression of Bacillus phytase gene (phy) in Escherichia coli and recovery of active enzyme from the inclusion bodies [J]. J Appl Microbiol,2008,105(4):1128-1137.
[120]Van Hartingsveldt M, Van Zwijl C M J, Harteveld G M, et al. Cloning, characeerization and overexpression of the phytase encoding gene (phyA) of Aspergillus niger [J]. Gene,1993,127:87-94.
[121]刘惠,杨杰.基于全序列比对相似度预测信号肽[J].上海交通大学学报,2008,42(1)：11-15.
[122]杜济良,赵洪亮.不同信号肽对胞外β-(1,3)-葡聚糖酶在巴斯德毕赤酵母中表达的影响[J].生物技术通报,2010,21(6)：798-802.
[123]侯静.甜菜硝酸还原酶基因克隆及生物信息学分析.硕士学位论文.黑龙江.东北农业大学.2008.
[124]Geourjon C, Deleage G SOPMA:significant improvements in protein secondary structure prediction by consensus prediction from multiple alignments [J]. Comput Appl Biosci.,1995,11 (6):681-684.
[125]Huiying L, Huoqing H. Peilong Y. et al. A Novel Phytase appA from Citrobacter amalonaticus CGMCC 1696: Gene Cloning and Overexpression in Pichia pastoris [J]. Curr Microbiol,2007,55:185-192.
[126]姚斌,范云六.植酸酶的分子生物学与基因工程[J].生物工程学报,2000,16(1)：1-5.
[127]许伟,仇明,余晓红等.芽孢杆菌植酸酶基因的克隆及生物信息学分析[J].食品科学,2011,32(7)：202-206.
[128]马东霞,詹志春等.GB/T 18634-2009.饲用植酸酶活性的测定分光光度法.北京：中国标准出版社,2009,8：1-6.
[129]秦兰霞,王利.玉米谷氨酰胺转胺酶基因的克隆及原核表达[J].食品科学,2010,31(3)：177-181.
[130]Shimizu M. Purification and characterization of phytase from Bacillus subtilis (natto) N-77 [J]. Biosci Biotechnol and Biochem,1992.56(8):1266-1269.
[131]Bae H D, Yanke L J, Cheng K J, et al. A novel staining method for detecting phytase activity [J]. J Microbiol Methods,1999,39(1):17-22.
[132]吴琪,段垒,谢红云等.中性植酸酶的酶学性质研究[J].饲料工业,2009,30(12)：15-18.
[133]Scorer C A, Clare J J, McCombie W R, et al. Rapid selection using G418 of high copy number transformants of Pichia pastoris for high level foreign gene expression [J]. Biotechnology,1994,12:181-184.
[134]崔志芳,刘娜,季爱云.植酸酶产生菌的快速筛选及发酵条件优化[J].中国酿造,2010,(7)：137-139.
[135]于平,陈益润.土壤中高产植酸酶芽孢杆菌菌株的筛选及鉴定[J].中国食品学报,2010,10(6)：116-121.
[136]Mullaney E J, Daly C B, Ullah A H. Advance in phytase research [J]. Adv Appl Microbiol,2004 (47): 157-199.
[137]王红宁,吴琦,赵海霞等.芽孢杆菌植酸酶基因在毕赤酵母中的分泌表达[J].浙江大学学报,2005,31(5)：621-627.
[138]Elkhalil E A, Maenner K, Borriss R, et al. In vitro and in vivo characteristics of bacterial phytases and their efficacy in broiler chickens [J]. Sci,2007,48 (2):64-70.
[139]Cregg J M, Cereghio J L, Shi J, et al. Recombinant protein expression in Pichia pastoris [J]. Mol Biotechnol, 2000,16(1):23-52.
[140]南泽吉三郎.栽桑学—基础与应用[M].东京：日本鸣风社出版,1984,299-326.
[141]刘先珍,朱建录,刘晓华.新型饲料源桑叶的营养价值及青贮技术[J].饲料工业,2005,26(23)：46-47.
[142]方小平,王转,陈茹梅等.能以植酸磷为唯一磷源生长的转基因甘蓝型油菜[J].作物学报,2010,36(2)：228-232.
[143]Yip W, Wang L J, Cheng C W, et al. The introduction of a phytase gene from Bacillus subtilis improved the growth performance of transgenic tobacco [J]. Biochemical and Biophysical Research Communications,2003, 310:1148-1154.
[144]Hegeman C E, Grabau E A. A novel phytase with sequence similarity to purple acid phosphatases is expressed in cotyledons of germinating soybean seedlings. Plant Physiol,2001,126:1598-1608.
[145]李春峰,黄科,甘进锋等.转植酸酶基因家蚕的制作及表达检测[J].昆虫学报,2009,52(7)：713-720.
[146]柯益富.桑树栽培及育种学[M].北京：中国农业出版社,1997,154-155.
[147]黄君霆,朱万民,向仲怀等.中国蚕丝大全[M].四川：四川科学技术出版社,1996,280-281.
[148]王茜龄,余茂德.桑子叶与胚轴不同区段离体再生植株的研究[J].蚕业科学,2005,31(3)：334-336.
[149]肖娅萍,胡雅琴,王枯之.卡那霉素对地灵愈伤组织诱导和生长的影响[J].西北植物学报,2003,23(2)：318-322.
[150]王丕武,张晓玲等.卡那霉素对大豆种子发芽的影响[J].吉林农业大学学报,1996,18(4)：8-21.
[151]傅荣昭.植物遗传转化技术手册[M].北京：中国科学技术出版社,1994.
[152]王勇.抗生索对桑树外植体生长与分化的影响[J].蚕业科学,1996,22(2)：72-76.
[153]王世杰,欧行奇.作物育种学总论[M].北京：中国农业科学技术出版社,2009,8.
[154]Hiroaki Machii. Leaf disk transformation of mulberry plant (Morus alba L.) by Agrobacterium Ti-plasmid [J]. J Seric Sct Jpn.1990,59(2):105-110.
[155]王彦文.桑树转基因受体系统的建立及遗传转化研究.博士学位论文.四川.四川农业大学.2006.
[156]张立军,梁宗锁.植物生理学[M].北京：科学出版社,2008,77-78.
[157]陆景陵.植物营养学[M].北京：中国农业大学出版社,2003,47-48.
[2]吴平,印莉萍,张立平等.植物营养分子生理学[M].北京：科学出版社,2001,102-103.
[3]呙于明,汤芹.植酸酶在养鸡业的应用研究(综述)[J].中国畜牧杂志,1997,33(6)：48-50.
[4]Purva Vats, Bharat Bhushan, U.C. Banerjee. Studies on the dephosphorylation of phytic acid in livestock feed using phytase from Aspergillus niger van Teighem [J]. Bioresource Technology,2009,100 (1):287-291.
[5]王红宁,黄勇,陈惠等.饲用微生物植酸酶的研究进展(综述)[J].国外畜牧学-饲料,1999,(3)：9-10.
[6]王红宁,吴琦等.黑曲霉N25植酸酶phvA基因的克隆及序列分析[J].微生物学报,2001,41(3)：310-314.
[7]Bergman E L, Autio K, Sanberg A S. Optimal conditions for paytate degradation, estimation of phytase activity, and localization of phytate in barley (cv. Blenheim) [J]. J Agric Food Chem,2000,48(10):4647-4655.
[8]赵玉生.植酸在食品工业中的应用[J].西部粮油科技,2000,25(6)：51-52.
[9]傅启高,李慧荃.三氯化铁比色法测植酸含量的研究[J].营养学报,1997,19(2)：210-220.
[10]刘巧茹,石起增,董文举.硝酸钍测定植酸含量方法的校正[J].平顶山学院学报,2006,21(5)：38-40.
[11]Dost K, Tokul O. Determination of phytic acid in wheat and wheat products by reverse phase high performance liquid chromatography [J]. Anal. Chim. Acta,2006,558 (1-2):22-27.
[12]肖传豪,陈来成.快速简便测定植酸的方法[J].化工时刊,2010,24(8)：66--68.
[13]Mason T J, Paniwnyk L, Lorimer J P. The uses of ultrasound in food technology [J]. Ultrasonic Chemistry, 1996,3(3):253-260.
[14]武雪芬,李玉贤,李桂兰.利用植酸对磺基水杨酸铁的脱色作用比色法测定植酸含量[J].河南科学,1996,14(1)：50-52.
[15]郑穗平.饲料工业酶技术[M].化学工业出版社,2008,8：29-30.
[16]吴谋成,袁俊华.植酸的毒理学评价和食用安全性[J].食品科学,1997,18(2)：46-49.
[17]Pallauf J, Rimbach G. Nutritional significance of phytic acid and Phytase [J]. Arch Tieremahr,1997,50(4): 301-319.
[18]黄君霆,朱万民,向仲怀等.中国蚕丝大全[M].四川科技出版社,1996,278-294.
[19]Boling S D, Douglas M W, Johnson M L, et al. The effects of dietary available phosphorus levels and phytase performance of young and older laying hens [J]. Poult Sci,2000,79:224-230.
[20]Cao L., Wang W., Yang C., et al. Application of microbial phytase in fish feed [J]. Enzyme and Microbial Technology,2007,40:497-507.
[21]Mallin M A. Impacts of industrial animal production on rivers and estuaries [J]. Am Sci,2000,88:26-37.
[22]Mullaney E. J, Daly C, et al. Advances in phytase esearch [J]. Adv Appl Microbiol,2000,47:157-199.
[23]Ullah A H, Sethumadhavan K. PhyA gene product of Aspergillus ficuum and Peniophora lycii produces dissimilar phytases [J]. Biochem. Biophys. Res. Commun.2003.303(2):463-468.
[24]Simons P C, Versteegh H A, Jongbloed A W. et al. Improvement of phosphorus availability by microbial phytase in broilers and pigs [J]. British Journal of Nutrition.1990.64(2):525-540.
[25]Reddy N R, Sathe S K, Salunkhe D K. Phytates in cereals and legumes [J]. Adv Food Res,1982,28:1-92.
[26]Kim Y O, Kim H K, Yu J H, et al. Cloning of the thermostable phytase gene (phy) from Bacillus sp.DS11 and its overexpression in Escherichia coli [J]. FEMS Microbiology Letters,1998,162:185-191.
[27]Ravindran V, Kornegay E T, Denbow D M, et al. Response of turkey poults to tiered levels of Natuphos phytase added to soybean mealbased semi-purified diets containing three levels of nonphytate phosphorus [J]. Poult Sci,1995,74:1843-1854.
[28]农业部办公厅：关于印发《饲用植酸酶使用指南》的通知,农办牧[2008]29号,中华人民共和国农业部公报,2008,6：37-38；
[29]Cromwell G L, Coffey R D, Parker G R, et al. Effiency of phytase in improving the bioavailability of phosphorus in soybean meal and corn-soybean meal diets for pigs [J]. J Anim Sci,1993,71:1831-1840.
[30]Vucenik L, Shamsuddin A M. Cancer inhibition by inositol Hexaphosphate (IP6) and inositol:From Laboratory to Clinic [J]. J. Nutr.,2003,133:3778-3784.
[31]Hieh T R, Ware J H. Survey of microorganisms for the production of extracellular phytase [J]. Appl. Microbiol., 1968,16:1348-1351.
[32]周庆安,姚军虎,何瑞国.植物性植酸酶及其应用[J].广东饲料,2004,4(14)：24-25.
[33]周庆安等.植物植酸酶减少养殖业磷排泄的研究[J].中国青年农业科学学术年报,2004,149-152；
[34]冯定远.酶制剂在饲料工业中的应用[M].北京：中国农业科学技术出版社,2005,11：299-309.
[35]Iqbal T H, Lewis K O. phytase activity in the human and rat small intestine [J]. Gut,1994,35(9):1233-1236.
[36]Ghareib M. Biosynthesis, Purification and Some Properties of Eatracellular Phytase from Aspergillus carneus [J]. Acta Microbiol. Hung.,1990,37(2):159-164.
[37]Wyss M, Pasamontes L, Remy R, et al. Comparison of the thermostability properties of three acid phosphatases from molds:Aspergillus fumigatus phytase, A. niger phytase and A. niger pH 2.5 acid phosphatase [J]. Appl. Environ. Microbiol.,1998,64:4446-4451.
[38]Han Y M, Roneker K R, et al. Adding wheat middling, microbial phytase, and citric acid to conr-soybean meal diets for growing pigs may replace inorganic phosphorus supplementation [J]. J. Anim. Sci.,1998,76: 2649-2656.
[39]Nakashima B A, Mcallister T A, Shanna R, et al. Diversity of phytase in the rumen [J]. Microb Ecol.,2007, 53(1):82-88.
[40]Schenk G, Guddat LW, Ge Y, et al. Identification of mammalian-like purple acid phoshpatases in a wide range of plants [J]. Gene,2000,250:117-125.
[41]Fu S, Sun J, Qian L, et al. Bacillus phytase:present scenario and future perspectives [J]. Appl Biochem Biotechnol,2008,151:1-8.
[42]Gylati H K, Chadha B S, Saini H S. Production and characterization of thermostable alkaline phytase from Bacillus laevolacticus isolated from rhizosphere soil [J]. J Ind Microbiol Biotechnol.,2007,34:91-98.
[43]Scott J J. A calcium-activated from pollen of Lilium longiflorum [J]. Plant Physiol,1986,82(1):333-335.
[44]Huang H, Shao N, Wang Y, et al. A novel beta-propeller phytase from Pedobacter nyacknsis MJ11 CGMCC 2503 with potential as an aquatic feed additive [J]. Appl Microbiol Biotechnol.2009.83:249-259.
[45]Lim B L, Yeung P, Cheng C, et al. Distribution and diversity of phytate-mineralizing bacteria [J]. ISME J.2007. 1:321-330.
[46]Choi Y M, Suh H J, Kim J M. Purification and properties of extracellular phytase from Bacillus sp. KHU-10 [J]. J Protein Chem,2001,20:287-292.
[47]Powar V K, Jagannathan V. Purification and properties of phytate-specific phosphatase from Bacillus subtilis [J]. J Bacteriol.1982,151:1102-1108.
[48]王亚茹,姚斌等.枯草芽孢杆菌中性植酸酶的纯化和酶学性质[J].微生物学报,2001,41(2)：198-203.
[49]Tye A J, Siu F K, Leung T Y, et al. Molecular cloning and the biochemical characterization of two novel phytases from B. subtilis 168 and B. licheniformis [J]. Appl Microbiol Biotechnol,2002,59:190-197.
[50]Ha N C, Oh B C, Shin S, et al. Crystal structures of a novel, themostable phytase in partially and fully calcium-loaded states [J]. Nature Struct Biol,2000(7):147-184.
[51]Kerovuo J, Lappalainen I, Reinikainen T. The metal dependence of Bacillus subtilis phytase [J]. Biochem Biophys Res Commun,2000,268:365-369.
[52]Shin S, Ha N C, Oh B C, et al. Enzyme mechanism and catalytic property of beta propeller phytase [J]. Structure,2001,9(9):851-858.
[53]Oh B C, Kim M H, Yun B S, et al. Ca(2+)-inositol phosphate chelation mediates the substrate specificity of beta-propeller phytase [J]. Biochemistry,2006,45(31):9531-9539.
[54]Greiner R, Farouk A, Alminger M L, et al. The pathway of dephosphorylation of myo-insitol hexakisphosphate by phytatedegeading enzymes of different Bacillus spp [J]. Can J Microbiol.,2002,48:986-994.
[55]彭景.烹饪营养学[M].中国纺织出版社,2008,68-69.
[56]林志勋,江世楷.禽畜消化道、肠道细菌和其消化能力之关系[J].中国畜牧杂志,1996,28(6)：17-27.
[57]姚斌,杨培龙,黄火清.中性植酸酶的研究与前景[J].生物产业技术,2010,4：42-47.
[58]伍喜林,陈贵才.应用动物营养学[M].四川科学技术出版社,2005,9：217-218.
[59]Lei X G, Porres J M, Mullaney E J, et al. Source, Structure and Application. Chapter 29, Industrial Enzyme Structure, Function and Application:Edited by Polaina J, MacCabe A.P.2007.
[60]Coello P, Maughan J P, Mendoza A, et al. Generation of low phytic acid Arabidopsis seeds expressing an E.coli phytase during embryo development [J]. Seed Science Research,2001,11 (1):185-291.
[61]Streb H, Irvine R F, Berridge M J, et al. Release of Ca2+ from a nonmitochondrial intracellular store in pancreatic acinar cells by inositol-1,4,5-trisphosphate [J]. Nature,1983,306:67-69.
[62]Claxon A, Morris C, Blake D, et al. The anti-inflammatorv effects of D-myo-inositol-1.2.6-trisphosphate (PP56) on animal models of inflammation [J]. Agents Actions.,1990,29:68-70.
[63]杨平平,许正宏,王燕等.微生物植酸酶进展[J].微生物学通报,2004,31(3)：111-115.
[64]王红艳,王云飞,王申涛.植酸酶的研究进展及应用[J].中国酿造,2010,4：24-25.
[65]pET System Manual.2003,3-5.
[66]Cregg J M. Vedvick T S, Raschke W C. Recent Advances in the expression of foreign genes in Pichia pastoris [J]. Bio/Technology.1993.11(8):905-910.
[67]Peng Y, Bu W, Kang L Y. Methylotrophic yeast system [J]. Biotechnol Information,2000,1:38-41.
[68]Zhuang S M, Landegent J E. Verschuerren C A. et al. Apoptin, a protein encoded by chicken anemia virus, induces cell death in human hematologic malignant cells [J]. Leukemia,1995,1:118-120.
[69]胡世界,罗素兰,张吉贞等.巴斯德毕赤酵母表达系统及其高水平表达策略[J].生物技术,2007,17(6)：78-83.
[70]Patricia S. Vary, Rebekka Biedendieek, Tobias Fuerch, et al. Bacillus megaterium from simple soil bacterium to industrial protein production host [J]. Applied Microbiology and Biotechnology,2007,76(5):957-967.
[71]Rygus T, Hillen W. Inducible high-level expression of heterologous genes in Bacillus megaterium using the regulatory elements of the xylose-utilization operon [J]. Applied Microbiology and Biotechnology,1991,35, 594-599.
[72]Wittchen K D, Meinhardt F. Inactivation of the major extracellular protease from Bacillus megaterium DSM319 by gene replacement [J]. Applied Microbiology and Biotechnology,1995,42(6):871-877.
[73]姚斌,袁铁铮,王元火等.来源于Bacillus的中性植酸酶基因的克隆及在大肠杆菌中的表达[J].生物工程学报,2001,17(1)：12-15.
[74]吴琦,王红宁,刘世贵等.枯草芽孢杆菌植酸酶基因在大肠杆菌中的表达及对酶热稳定性的影响[J].高技术通讯,2004,14(5)：23-27.
[75]邹立扣,王红宁,吴琦等.植酸酶phyC基因表达载体的构建及其在E.coli中的表达[J].生物技术,2004,14(3)：11-14,
[76]牟琳.枯草芽孢杆菌WHNB02中性植酸酶基因在巨大芽孢杆菌中的表达研究.硕士学位论文.四川.四川农业大学.2008.
[77]Guerrero-Olazaran M, Rodriguez-Blanco L, Carreon-Trevino J G, et al. Expression of a Bacillus Phytase C Gene in Pichia pastoris and Properties of the Recombinant Enzyme [J]. Appl. Environ. Microbiol.,2010, 76(16):5601-5608.
[78]Zou L K, Wang H L, Pan X, et al. Design and Expression of a Synthetic phyC Gene Encoding the Neutral Phytase in Pichia pastoris [J]. Acta Biochimica et Biophysica Sinica,2006,38(11):803-811.
[79]Larrick J W, Thomas D W. Producing proteins in transgenic plants and animals [J]. Curr. Opin. Biotechnol., 2001,12:411-418.
[80]Pen J, Verwoerd T C, van Paridon P A, et al. Phytase-containing transgenic seeds as anovel feed additive for improved phosphorus utilization [J]. Bio/Technology.,1993,11:811-814.
[81]Verwoerd T C, Van Paridon P A, Van Ooyen A J J, et al. Stable accumulation of Aspergillus niger phytase in transgenic tobacco leaves [J]. Plant Physiol.1995,109:1199-1205.
[82]Ullah A H J, Sethumadhavan K, Mullaney E J, et al. Characterization of recombinant fungal phytase (phyA) expressed in tobacco leaves [J]. Biochem Biophys Res Commun.,1999,264:201-206.
[83]Mudge S R, Smith F W, Richardson A E. Root-specific and phosphate-regulated expression of phytase under the control of a phosphate tr ansporter promoter enables Arabidopsis to grow on phytate as a sole P source [J]. Plant Science,2003,165:871-878.
[84]Gutknecht K. Green genes:alfalfa biofanning is about to take root [J]. Wise Agrict.,1997,3:8-10.
[85]Brinch-Pedersen H, Olesen A, Rasmussen S K, et al. Generation of transgenic wheat (Triticum aestivum L.) for constitutive accumulation of an Aspergillus phytase [J]. Mol Breed..2000.6:195-206.
[86]Ponstein A S, Bade J B, Verwoerd T C, et al. Stable expression of Phytase (phyA) in canola (Brassica napus) seeds:towards a commercial product [J]. Mol Breed.,2002,10:31-44.
[87]Denbow D M, Grabau E A, Lacy G H, et al. Soybeans transformed with a fungal phytase gene improve phosphorus availability for broilers [J]. Poultry Sci.,1998,77:878-881.
[88]Zhang Z B, Kornegay E T, Radcliffe J S, et al. Comparison of genetically engineered microbial and plant phytase for young broilers [J]. Poultry Sci.,2000,79:709-717.
[89]Hong C, Cheng K, Tseng T, et al. Production of two highly active bacterial phytases with broad pH optima in germinated transgenic rice seeds [J]. Transgenic Res.,2004,13:29-39.
[90]Xiao K, Harrison M, Wang Z Y. Transgenic expression of a novel M.truncatula phytase gene results in improved acquisition of organic phosphorus by Arabidopsis [J]. Planta.,2005,222:27-36.
[91]Xiao K, Zhang J H, Harrison M, et al. Ectopic expression of a phytase gene from Medicago truncatula Barrel Medic enhances phosphorus absorption in plants [J]. Journal Integrative Plant Biology.,2006,48(1):35-43.
[92]Li J, Hegeman C E, Hanlon R W, et al. Secretion of active recombinant phytase from soybean cell suspention cultures [J]. Plant Physiol.,1997,114:1103-1111.
[93]Austin S, Bingham E T, et al. Production and field performance of transgenic alfalfa(Medicago sativa L.) expressing alpha-amylase and manganese-dependent lignin peroxidase [J]. Euphytica.,1995,85:381-393.
[94]Ponstein A S, Bade J B, et al. Stable expression of Phytase (phyA) in canola (Brassica napus) seeds:towards a commercial product [J]. Molecular Breeding.,2002,10:31-44.
[95]Chen R M, Yao B, Fan Y L, et al. Transgenic maize plants expressing a fungal phytase gene [J]. Transgenic Res.,2008,17:633-643.
[96]范云六：利用玉米种子生物反应器成功培育出生产高活性植酸酶并稳定遗传的转基因玉米,新华网北京2007年9月10日电,记者姚润丰.
[97]谈建中,楼程富,钟名其等.大豆球蛋白基因表达载体的构建及对桑树的遗传转化[J].蚕业科学,1999,25(1)：6-11.
[98]陆小平,楼成富,王波等.用植株转化法将GUS基因导入桑树幼苗的研究[J].蚕业科学,2004,30(2)：120-132.
[99]Shalini L, Vibha G, Paramjit K. Overexpression of HVA1 gene from barley generates tolerance to salinity and water stress in transgenic mulberry (Morus indica) [J]. Transgenic Res.,2008,17:651-663.
[100]侯小峰.植酸酶之困[J].中国畜牧杂志,2008,44(8)：1-5.
[101]Greiner R, Carlson NG, Alminger M L. Stereospecificity of myo-inositol hexa phosphate dephosphorylation by phytate degrading enzyme by Escherichia coli [J]. J Biotech,2000,84:53-62.
[102]Purva V, Uttam C Banerje. Production studies and catalytic properties of phytases (myo-inositolhexakisphos-phate phosphohydrolases):an overview. Enzyme and Microbial Technology,2004,35(1):3-14.
[103]Afnah S, Yazid A M, Anis Shobirin M H, et al. Phytase:application in food industry. International Food Research Journal,2010,17:13-21.
[104]柯益富.桑树栽培及育种学[M].北京：中国农业出版社,1997：156-159.
[105]Maenz D D. Classen H L. Phytase activity in the small intestinal brush-border membrane of the chicken [J]. Poult Sci.1998,77:557-563.
[106]Naqvi S W A. Jayakumar D A, Narvekar P V, et al. Increased marine production of N2O due to intensifying anoxia on the Indian continental shelf [J]. Nature,2000,408:346-349.
[107]陆欣主编.十壤肥料学[M].北京：中国农业大学出版社,2002,208-227.
[108]Saad N. Esa N M. Ithnin H, et al. Optimization of optimum condition for phytic acid extraction from rice bran [J]. African Journal of Plant Science,2011,5(3):168-176.
[109]Kwanyuen P, Burton J W. A simple and rapid procedure for phytate determination in soybeans and soy products [J]. J Am Oil Chem Soc,2005,82(2):81-85.
[110]余安,王承明.超声辅助提取花生粕中植酸工艺优化[J].食品科学,2010,31(10)：179-183.
[111]沈维治,廖森泰,刘吉平等.用二次回归正交旋转组合设计优化桑叶多酚的提取工艺[J].蚕业科学,2009,35(3)：594-598.
[112]Camire A L, Clydesdale F M. Analysis of phytic acid in foods by HPLC [J]. J Food Sci,1982,47(2): 575-578.
[113]王忠.植物生理学[M].北京：中国农业出版社,2000：84-85.
[114]李朝霞,王爱民,李小敏.中性植酸酶高产菌株的筛选及产酶条件研究[J].微生物学通报,2007,34(4)：633-641.
[115]霍春雁,李锋,单冉东.生物化学实验方法和技术[M].北京：科学出版社.2002.
[116]吴琦.枯草芽孢杆菌植酸酶phyC基因的克隆及其表达研究[D].成都：四川大学,2004.
[117]陈艳,孙建义,赵学新等Bacillus amyloliquefaciens中性植酸酶基因的原核表达及蛋白纯化和性质[J].食品与生物技术学报,2005,24(2)：60-65.
[118]Chen X H, et al. Comparative analysis of the complete genome sequence of the plant growth-promoting bacterium Bacillus amyloliquefaciens FZB42 [J]. Nat Biotechnol,2007,25(9):1007-1014.
[119]Rao D E, Rao K. V, Reddy V D. Cloning and expression of Bacillus phytase gene (phy) in Escherichia coli and recovery of active enzyme from the inclusion bodies [J]. J Appl Microbiol,2008,105(4):1128-1137.
[120]Van Hartingsveldt M, Van Zwijl C M J, Harteveld G M, et al. Cloning, characeerization and overexpression of the phytase encoding gene (phyA) of Aspergillus niger [J]. Gene,1993,127:87-94.
[121]刘惠,杨杰.基于全序列比对相似度预测信号肽[J].上海交通大学学报,2008,42(1)：11-15.
[122]杜济良,赵洪亮.不同信号肽对胞外β-(1,3)-葡聚糖酶在巴斯德毕赤酵母中表达的影响[J].生物技术通报,2010,21(6)：798-802.
[123]侯静.甜菜硝酸还原酶基因克隆及生物信息学分析.硕士学位论文.黑龙江.东北农业大学.2008.
[124]Geourjon C, Deleage G SOPMA:significant improvements in protein secondary structure prediction by consensus prediction from multiple alignments [J]. Comput Appl Biosci.,1995,11 (6):681-684.
[125]Huiying L, Huoqing H. Peilong Y. et al. A Novel Phytase appA from Citrobacter amalonaticus CGMCC 1696: Gene Cloning and Overexpression in Pichia pastoris [J]. Curr Microbiol,2007,55:185-192.
[126]姚斌,范云六.植酸酶的分子生物学与基因工程[J].生物工程学报,2000,16(1)：1-5.
[127]许伟,仇明,余晓红等.芽孢杆菌植酸酶基因的克隆及生物信息学分析[J].食品科学,2011,32(7)：202-206.
[128]马东霞,詹志春等.GB/T 18634-2009.饲用植酸酶活性的测定分光光度法.北京：中国标准出版社,2009,8：1-6.
[129]秦兰霞,王利.玉米谷氨酰胺转胺酶基因的克隆及原核表达[J].食品科学,2010,31(3)：177-181.
[130]Shimizu M. Purification and characterization of phytase from Bacillus subtilis (natto) N-77 [J]. Biosci Biotechnol and Biochem,1992.56(8):1266-1269.
[131]Bae H D, Yanke L J, Cheng K J, et al. A novel staining method for detecting phytase activity [J]. J Microbiol Methods,1999,39(1):17-22.
[132]吴琪,段垒,谢红云等.中性植酸酶的酶学性质研究[J].饲料工业,2009,30(12)：15-18.
[133]Scorer C A, Clare J J, McCombie W R, et al. Rapid selection using G418 of high copy number transformants of Pichia pastoris for high level foreign gene expression [J]. Biotechnology,1994,12:181-184.
[134]崔志芳,刘娜,季爱云.植酸酶产生菌的快速筛选及发酵条件优化[J].中国酿造,2010,(7)：137-139.
[135]于平,陈益润.土壤中高产植酸酶芽孢杆菌菌株的筛选及鉴定[J].中国食品学报,2010,10(6)：116-121.
[136]Mullaney E J, Daly C B, Ullah A H. Advance in phytase research [J]. Adv Appl Microbiol,2004 (47): 157-199.
[137]王红宁,吴琦,赵海霞等.芽孢杆菌植酸酶基因在毕赤酵母中的分泌表达[J].浙江大学学报,2005,31(5)：621-627.
[138]Elkhalil E A, Maenner K, Borriss R, et al. In vitro and in vivo characteristics of bacterial phytases and their efficacy in broiler chickens [J]. Sci,2007,48 (2):64-70.
[139]Cregg J M, Cereghio J L, Shi J, et al. Recombinant protein expression in Pichia pastoris [J]. Mol Biotechnol, 2000,16(1):23-52.
[140]南泽吉三郎.栽桑学—基础与应用[M].东京：日本鸣风社出版,1984,299-326.
[141]刘先珍,朱建录,刘晓华.新型饲料源桑叶的营养价值及青贮技术[J].饲料工业,2005,26(23)：46-47.
[142]方小平,王转,陈茹梅等.能以植酸磷为唯一磷源生长的转基因甘蓝型油菜[J].作物学报,2010,36(2)：228-232.
[143]Yip W, Wang L J, Cheng C W, et al. The introduction of a phytase gene from Bacillus subtilis improved the growth performance of transgenic tobacco [J]. Biochemical and Biophysical Research Communications,2003, 310:1148-1154.
[144]Hegeman C E, Grabau E A. A novel phytase with sequence similarity to purple acid phosphatases is expressed in cotyledons of germinating soybean seedlings. Plant Physiol,2001,126:1598-1608.
[145]李春峰,黄科,甘进锋等.转植酸酶基因家蚕的制作及表达检测[J].昆虫学报,2009,52(7)：713-720.
[146]柯益富.桑树栽培及育种学[M].北京：中国农业出版社,1997,154-155.
[147]黄君霆,朱万民,向仲怀等.中国蚕丝大全[M].四川：四川科学技术出版社,1996,280-281.
[148]王茜龄,余茂德.桑子叶与胚轴不同区段离体再生植株的研究[J].蚕业科学,2005,31(3)：334-336.
[149]肖娅萍,胡雅琴,王枯之.卡那霉素对地灵愈伤组织诱导和生长的影响[J].西北植物学报,2003,23(2)：318-322.
[150]王丕武,张晓玲等.卡那霉素对大豆种子发芽的影响[J].吉林农业大学学报,1996,18(4)：8-21.
[151]傅荣昭.植物遗传转化技术手册[M].北京：中国科学技术出版社,1994.
[152]王勇.抗生索对桑树外植体生长与分化的影响[J].蚕业科学,1996,22(2)：72-76.
[153]王世杰,欧行奇.作物育种学总论[M].北京：中国农业科学技术出版社,2009,8.
[154]Hiroaki Machii. Leaf disk transformation of mulberry plant (Morus alba L.) by Agrobacterium Ti-plasmid [J]. J Seric Sct Jpn.1990,59(2):105-110.
[155]王彦文.桑树转基因受体系统的建立及遗传转化研究.博士学位论文.四川.四川农业大学.2006.
[156]张立军,梁宗锁.植物生理学[M].北京：科学出版社,2008,77-78.
[157]陆景陵.植物营养学[M].北京：中国农业大学出版社,2003,47-48.