转植酸酶-抗菌肽融合基因水稻BPL9K的植酸酶活性分析
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摘要
本研究检测了转植酸酶-抗菌肽融合基因水稻BPL9K及其杂交组合种子的植酸酶活性。结果表明:(1) 3个转化体的植酸酶活性差异极显著(p<0.01),BPL9K-1植酸酶活性最高,平均为28.82 U/g,BPL9K-4次之,平均为25.77 U/g,BPL9K-2最低,平均为17.11 U/g;各转化体的植酸酶活性均能稳定遗传;纯合体和杂合体间植酸酶活性差异不显著(p>0.05)。(2)植酸酶基因表达显著受遗传背景和栽培条件的影响,3个转化体盆栽的植酸酶活性均极显著高于大田(p<0.01);不同杂交组合的植酸酶活性差异显著(p<0.05),最高达到20.01 U/g,最低只有8.59 U/g。(3)常温储存6个月,BPL9K-1和BPL9K-2的植酸酶活性下降均不显著(p>0.05),但BPL9K-4的植酸酶活性均显著下降(p<0.05);常温贮存12个月,3个转化体的种子植酸酶活性均显著降低(p<0.05),但仍保持较高的植酸酶活性。证明在适宜条件下BPL9K可以进行较长时间的保存。本研究结果为高植酸酶活性水稻品种培育提供了技术支撑。
This study detected the phytase activity of genetically modified rice BPL9 K containing phytase-lfcin fusion gene(PhyLf) and its hybrid combinations, and concluded as follows:(1) Phytase activities were significantly different among three transformants(p <0.01), BPL9 K-1 had the highest phytase activity with an average of28.82 U/g, BPL9 K-4 came to the second with an average of 25.77 U/g, and BPL9 K-2 was the lowest with an average of 17.11 U/g. The phytase activity of all the transformants could stably inherit. The phytase activities were not significantly different between homozygous and heterozygous genotype(p>0.05).(2) The expression of PhyLf gene was significantly affected by cultivation conditions and genetic background. Phytase activities of rice planted in pot were significantly higher than those in paddy(p<0.01). Different hybrid combinations had significantly different phytase activities(p<0.05), the highest up to 20.01 U/g on average and the lowest low to 8.59 U/g on average.(3) After storing at room temperature for six months, the phytase activities of BPL9 K-1 and BPL9 K-2 did not decrease significantly, but that of BPL9 K-4 decreased significantly(p<0.05). After storing at room temperature for twelve months, the phytase activities in seeds of three transformants decreased significantly(p<0.05), although all the three transformants were still keeping high phytase activities. That suggested the phytase activities in seeds of genetically modified rice BPL9 K could be maintained for a long time under suitable storage condition. This study might provide technical support for the breeding of rice varieties with high phytase activity.
This study detected the phytase activity of genetically modified rice BPL9 K containing phytase-lfcin fusion gene(PhyLf) and its hybrid combinations, and concluded as follows:(1) Phytase activities were significantly different among three transformants(p <0.01), BPL9 K-1 had the highest phytase activity with an average of28.82 U/g, BPL9 K-4 came to the second with an average of 25.77 U/g, and BPL9 K-2 was the lowest with an average of 17.11 U/g. The phytase activity of all the transformants could stably inherit. The phytase activities were not significantly different between homozygous and heterozygous genotype(p>0.05).(2) The expression of PhyLf gene was significantly affected by cultivation conditions and genetic background. Phytase activities of rice planted in pot were significantly higher than those in paddy(p<0.01). Different hybrid combinations had significantly different phytase activities(p<0.05), the highest up to 20.01 U/g on average and the lowest low to 8.59 U/g on average.(3) After storing at room temperature for six months, the phytase activities of BPL9 K-1 and BPL9 K-2 did not decrease significantly, but that of BPL9 K-4 decreased significantly(p<0.05). After storing at room temperature for twelve months, the phytase activities in seeds of three transformants decreased significantly(p<0.05), although all the three transformants were still keeping high phytase activities. That suggested the phytase activities in seeds of genetically modified rice BPL9 K could be maintained for a long time under suitable storage condition. This study might provide technical support for the breeding of rice varieties with high phytase activity.
引文
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Li X.H.,Liu Y.,Yan R.Y.,Kong Y.B.,Li G.L.,Chang W.S.,and Zhang C.Y.,2013,Introducing phytase gene phyA into different soybean varieties from transgenic line utilizing sexual hybridization,Zhiwu Yichuan Ziyuan Xuebao(Journal of Plant Genetic Resources),14(1):124-131(李喜焕,刘渊,闫瑞叶,孔佑宾,李桂兰,常文锁,张彩英,2013,杂交转育植酸酶phyA大豆阳性材料筛选研究,植物遗传资源学报,14(1):124-131)
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Van Gorcom R.F.M.,Van Hartingsveldt W.,Van Paridon P.A.,Veenstra A.E.,Luiten R.G.,and Selten G.C.M.,1996,Cloning and expression of phytase from Aspergillus,Biotechnology Advances,14(4):453
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Wang H.Y.,Tang H.O.,Wang X.R.,Yang L.L.,and Liu Y.,2013,Assessments of the thermostability of thermostable phytase,Siliao Gongye(Feed Industry),34(20):15-18(王海燕,汤海鸥,王晓睿,杨禄良,刘影,2013,耐高温植酸酶的热稳定性评估方法分析,饲料工业,34(20):15-18)
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Cao G.C.,Wu D.B.,Chen H.Q.,Qiang X.L.,Dong M.,Kou H.,Wang J.L.,Hou L.B.,and Li M.,2004,Relationship between temperature,sunshine and quality of spring-sowing wheat,Zhongguo Nongye Kexue(Scientia Agricultura Sinica),37(5):663-669(曹广才,吴东兵,陈贺芹,强小林,冬梅,寇皞,王建林,侯立白,李萌,2004,温度和日照与春播小麦品质的关系,中国农业科学,37(5):663-669)
Chen R.,Xue G.,Chen P.,Yao B.,Yang W.Z.,Ma Q.L.,Fan Y.L.,Zhao Z.Y.,Mitchell C.,and Tarczynski S.J.R.,2008,Transgenic maize plants expressing a fungal phytase gene,Transgenic Research,17(4):633-643
Dai X.H.,Wang S.J.,and Cao H.X.,2005,Effect of ecological condition and culture techniques on the yield and protein content of spring wheat,Nongye Kexue Yanjiu(Journal of Agricultural Sciences),26(4):7-11(代晓华,王世敬,曹宏鑫,2005,生态条件和栽培技术措施对春小麦产量和蛋白质含量的影响,农业科学研究,26(4):7-11)
Denbow D.,Grabau E.,Lacy G.,Kornegay E.,Russell D.,and Umbeck P.,1998,Soybeans transformed with a fungal phytase gene improve phosphorus availability for broilers,Poultry Science,77(6):878-881
Deng F.,Wang L.,Ye D.C.,Ren W.J.,and Yang W.Y.,2012,Effects of ecological conditions and cultivation methods on rice starch RVA profile characteristics and protein content,Zuowu Xuebao(Acta Agronomica Sinica),38(4):717-724(邓飞,王丽,叶德成,任万军,杨文钰,2012,生态条件及栽培方式对稻米RVA谱特性及蛋白质含量的影响,作物学报,38(4):717-724)
Gao C.Q.,Ma Q.G.,Zhao L.H.,Zhang J.,and Ji C.,2014,Effect of dietary phytase transgenic corn on physiological characteristics and the fate of recombinant plant DNA in laying hens,Asian-Australasian Journal of Animal Sciences,27(1):77
George T.S.,Richardson A.E.,Hadobas P.A.,and Simpson R.J.,2004,Characterization of transgenic Trifolium subterraneum L.which expresses phyA and releases extracellular phytase:growth and P nutrition in laboratory media and soil,Plant Cell and Environment,27(11):1351-1361
Gontia I.,Tantwai K.,Rajput L.P.S.,and Tiwari S.,2012,Transgenic plants expressing phytase gene of microbial origin and their prospective application as feed,Food Technology and Biotechnology,50(1):3-10
Hong C.Y.,Cheng K.J.,Tseng T.H.,Wang C.S.,Liu L.F.,and Yu S.M.,2004,Production of two highly active bacterial phytases with broad pH optima in germinated transgenic rice seeds,Transgenic Research,13(1):29-39
Li G.L.,Yang S.H.,Li M.G.,Qiao Y.K.,and Wang J.H.,2009,Functional analysis of an Aspergillus ficuum phytase gene in Saccharomyces cerevisiae and its root-specific,secretory expression in transgenic soybean plants,Biotechnology Letters,31(8):1297-1303
Li J.,Hou H.J.,Huang F.S.,Xia X.J.,and Xiao G.Y.,2008,Transformation of phytase gene into rice by particle bombardment,Yingyong Yu Huanjing Shengwu Xuebao(Chinese Journal of Applied and Environmental Biology),14(1):6-10(李进,侯海军,黄复深,夏新界,肖国樱,2008,基因枪介导获得转植酸酶基因水稻研究,应用与环境生物学报,14(1):6-10)
Li Q.F.,Liu Q.Q.,Zhang D.J.,Wang H.M.,Yu H.X.,Gu M.H.,and Yao H.Q.,2006,Expression of recombinant phytase in transgenic rice,Zhongguo Shuidao Kexue(Chinese Journal of Rice Science),20(3):243-247(李钱峰,刘巧泉,张达江,王红梅,于恒秀,顾铭洪,姚泉洪,2006,转基因水稻中重组植酸酶的表达,中国水稻科学,20(3):243-247)
Li X.H.,Liu Y.,Yan R.Y.,Kong Y.B.,Li G.L.,Chang W.S.,and Zhang C.Y.,2013,Introducing phytase gene phyA into different soybean varieties from transgenic line utilizing sexual hybridization,Zhiwu Yichuan Ziyuan Xuebao(Journal of Plant Genetic Resources),14(1):124-131(李喜焕,刘渊,闫瑞叶,孔佑宾,李桂兰,常文锁,张彩英,2013,杂交转育植酸酶phyA大豆阳性材料筛选研究,植物遗传资源学报,14(1):124-131)
Liu M.Y.,Peng J.,Liu M.Y.,and Gao Y.,2007,Research progress in improving phytase thermostability by post-processing technics,Siliao Gongye(Feed Industry),28(11):1-3(刘梅英,彭健,刘敏跃,高雁,2007,从后处理工艺提高植酸酶热稳定性的研究进展,饲料工业,28(11):1-3)
Ma X.,2001,Study on the applied properties of plant phytase,Thesis for M.S.,Northeast Agricultural University,Supervisor:Shan A.S.,pp.26-27(马玺,2001,植物植酸酶应用特性的研究,硕士学位论文,东北农业大学,导师:单安山,pp.26-27)
Mu J.P.,2005,Effect of different sowing dates on quality characters of spring wheat and synthesis and accumulation of storage protein,Thesis for M.S.,Gansu Agricultural University,Supervisor:Shang X.W.,pp.26-27(慕军鹏,2005,不同播期对春小麦品质性状及其贮藏蛋白合成和积累的影响,硕士学位论文,甘肃农业大学,导师:尚勋武,pp.25-39)
Naqvi S.,Ramessar K.,FarréG.,Sabalza M.,Miralpeix B.,Twyman R.M.,Capell T.,Zhu C.F.,and Christou P.,2011,High-value products from transgenic maize,Biotechnology Advances,29(1):40-53
Pasamontes L.,Haiker M.,Wyss M.,Tessier M.,and van Loon A.P.,1997,Gene cloning,purification,and characterization of a heat-stable phytase from the fungus Aspergillus fumigatus,Applied and Environmental Microbiology,63(5):1696
Pen J.,Verwoerd T.C.,Paridon P.A.V.,Beudeker R.F.,Elzen P.J.M.V.D.,Geerse K.,Klis J.D.V.D.,Versteegh H.A.J.,Ooyen A.J.J.V.,and Hoekema A.,1993,Phytase-containing transgenic seeds as a novel feed additive for improved phosphorus utilization,Biotechnology,11(7):811-814
Shen Y.O.,Peng H.W.,and Pan G.T.,2005,Research progress in transgenic plants expressing recombinant phytase,Zhongguo Shengwu Gongcheng Zazhi(China Biotechnology),25(1):29-32(沈亚欧,彭焕伟,潘光堂,2005,转基因植物表达植酸酶研究进展,中国生物工程杂志,25(1):29-32)
Suzuki U.,Yoshimura K.,and Takaishi M.,1907,Ueber ein enzym"Phytase"das"Anhydro-oxy-methylen diphosphor saure"spaltet,Tokyo Imper.Univ.Coll.Agric.Bull.,(7):503-512
Van Gorcom R.F.M.,Van Hartingsveldt W.,Van Paridon P.A.,Veenstra A.E.,Luiten R.G.,and Selten G.C.M.,1996,Cloning and expression of phytase from Aspergillus,Biotechnology Advances,14(4):453
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