摘要
硒是人及哺乳动物必需的一种微量元素。硒参与甲状腺激素代谢、抗氧化系统和机体免疫功能的正常发挥。在原核生物、真核生物和古细菌中都发现有硒蛋白的存在,其中哺乳动物硒蛋白现已发现近30种。但是,植物硒蛋白的研究相对滞后,迄今为止仅见于衣藻硒蛋白的报道。高等植物中硒蛋白是否存在的问题,迄今尚未有明确答案。因此应用生物信息学的研究手段,对高等植物中可能存在的硒蛋白线索进行找寻和分析,在理论研究和实际应用中都具有重要意义。
本文第一部分综合总结归纳了硒蛋白领域的研究方法和相关成果,对目前植物硒蛋白研究停滞不前的原因进行了分析。在此基础上,精心设计研究路线和方法,选取硬粒小麦Durum Wheat为研究对象,综合利用生物信息学手段,对硬粒小麦Durum Wheat中可能存在的硒蛋白进行了探索性研究。
鉴于目前已发现的硒蛋白均具有SECIS特征结构,首先对Genbank中的Durum Wheat表达核酸序列进行逐一筛选,找到其具有SECIS的序列,共发现47个具有SECIS特征结构的目标序列。通过进一步分析和筛选,确定以数据量完整的AY146587.1序列及其互补序列为进一步的研究目标。
应用Vector NT等工具对目标序列和衣藻硒蛋白进行综合比较分析,最终在目标序列发现一段与衣藻硒蛋白具有较高的结构相似性的表达区段,并在此基础上研究分析该目标序列编码硒蛋白(或同源物)的可能性。针对上述表达区段没有发现TGA碱基区的问题,再次对目标序列进行了更为全面的搜索和分析研究,又发现另一段同时具有与衣藻硒蛋白相似结构特征和TGA碱基区的序列段。这2段表达区位置临近并且有交集,都位于GENSCAN预测得到的基因段内。
将上述2段序列作为目标对象,以其翻译后氨基酸序列的结构特征为突破口,利用现有蛋白质数据库和分析工具进行了大量的研究分析,发现了具有意义的生物信息线索,这些线索均显示目标序列可能具有和动物硒蛋白相似的功能。综合分析上述研究结果,认为在目标序列中这一表达区段范围内,存在硒蛋白的可能性很大,并推测其功能作用与硬粒小麦Durum Wheat胚胎发育时期能量调节相关。
同时在研究过程中发现,完全依赖基于现有知识的生物信息学分析工具和方法对高等植物可能具有的硒蛋白进行研究,局限性很大,这与动物硒蛋白研究领域的情况有很大不同。结合本课题研究的结果,综合分析目前植物硒蛋白研究领域现状,提出合理的解释和推断:高等植物中存在功能与动物硒蛋白相似的硒蛋白,但其具有与动物硒蛋白不同的编码机理,甚至不能排除高等植物中存在未知氨基酸的可能性。正是由于这个原因,导致了在硒蛋白研究成果领域中动物和植物的迥然不同。
本论文第一部分阐述的研究成果一方面在高等植物硒蛋白研究领域中取得有意义的发现和突破;另一方面也提出了大胆的设想和推测,为今后高等植物硒蛋白研究提供了新的思路和方法。
本文第二部分主要针对当前生物试验中高通量筛选的实际需要,同时也考虑到今后植物硒蛋白生物验证试验中可能出现的高通量筛选需求,利用图论方法,建立了一个高效准确的筛选策略模型,并编制程序实现。通过分析,确定了最佳分组大小与所需次数及提高效率的关系,并对该策略模型进行生物学验证,从而建立了一种准确快速的筛选方法。该筛选方法相对于单个检测速度能提高10-100倍。该方法在具体操作中可根据实际情况灵活处理,通过调整分组大小,利用程序计算出需要检测次数的上界,作为实验的有利参考。利用这种方法能在92次PCR次数之内从1000个植株材料中快速准确地筛选出10个阳性的转基因植株。此方法为植物基因工程大量无标记基因转化后代的快速筛选提供了条件,并对涉及从M个样品中寻找N个目标的实验有一定的指导作用。
Selenium is a microelement which is essential to mammalian and hunman being. It participates process of the metabolism of thyroid gland,antioxidant system and ensuring organism exert normal immune function. Selenoproteins were found in eukaryotes and archaea. However, no selenoprotein was found in plants except the Chlamydomonas. There is no clear answer whether the selenoproteins exist in plants until now. So it is important to search and research the clues of the possible selenoproteins in plants with bioinformatics means.
The first part of this paper synthetically sumed up the research means and outcomes in the area of selenoproteins research, the reason for the stagnant situation of research of selenoproteins in plants was analysed also. At the basis of previous analysis, we choosed the Durum Wheat as the research target and carefully designed the research methods. The bioinformatics research of the target was carried out subsequently.
Due to the selenoproteins having the SECIS, we searched and filtered the expressed nucleic acid sequences of Durum Wheat from Genbank to find out the objects with SECIS and 47 object sequences was found aggregately. The AY146587.1 sequence ( with complementary sequence of AY146587.1 sequence)was determined the research object finally for its sufficient data.
An expressed area in object sequence was found which had highly similar structure with the selenoproteins of Chlamydomonas after the object sequence and six selenoproteins of Chlamydomonas were synthetically comparative analysed with tools such as Vector NT. Subsequently, the possibility, that whether the object sequence coding the selenoprotein, has been analysed. Aiming at the query that there was no TGA in the above-mentioned sequence, more full-scale searching work was made and another expressed sequence which has TGA and the similar structural character with the selenoproteins of Chlamydomonas was found. The two expressed sequences located in the same gene area presumed by GENSCAN and were contiguous to each other.
Whlie the two object expressed sequences were translated, much researching and analysing work was carried out. Some clues were found and which revealed the object expressed sequences had the similar function characters with the selenoproteins. Due to all-inclusive result, we presumed the area where the object sequences located had high possibility to coding the selenoproteins which had relationg to enerye regulation in durum wheat embryo growth period.
In course of our work, we found it was hard to apply the existing tools and methods which were builted on the basis of known knowledge to the research work to the possible selenoproteins in plants. It was so different from the area of a creatural selenoproteins. One reasonable predication was put forward that there were selenoproteins in plants which had similar functions to the creatural selenoproteins but different coding principle whereas. We even predicated that there may be unknown amino acid existed.
All the content in the first part of this paper had described the important result of our research work in selenoprotein of plants. At the same time, innovative thinking and method was put forward to promote the studying of selenoprotein in plants.
On the other hand, an optimal pooling system called Accurate and Fast Target Screening has been developed for high-throughput identifying the rare marker-free transformants in plant genetic engineering and which may be important for high-throughput identifying test of the selenoproteins in plants for the future. This system could identify targets between ten- and hundred-fold more efficiently than analysis of individual samples. By considering the efficiency for different values of the proportion and corresponding optimal group size for varying total numbers of samples, we are able to estimate an upper limit for the number of tests that are required. The application of this system to determine the transgene in an artificially constructed population of transgenic and non-transgenic wheat lines successfully identified the 10 positive samples located randomly within 1000 samples using only 92 PCR reactions. The same approach was also applied to determine transgene expression by SDS-PAGE of seed proteins. This system should be applied to high-throughput screening for rare marker-free transformants in plant genetic engineering.
引文
[1]刘晓琰,施安国.微量元素硒的研究进展.中国临床药理杂志,2001,17(5):375~378
[2] Taylor EW, Nadimpalli RG, Ramanathan CS. Genomic structures of viral agents in relation to the biosynthesis of selenoproteins. Biol Trace Elem Res, 1997,56: 63~91
[3] Look MP,Rockstroh JK,Rao GS,et al ,Serum selenium versus lymphocyte subsets and markers of disease progression and inflammatory response in human immunodeficiency virus-infection.Biol Trace Elem Res, 1997, 56: 31~41
[4] Baum MK, Shor-Posner G,Lai S,et al. High risk of HIV-related mortality is associated with selenium deficiency.J Acquir Immune Defic Syndr, 1997, 15: 370~374
[5] Yu SY,Zhu YJ,Li WG . Protective role of selenium against hepatitis B virus and primary liver cancer in Qidong.Biol Trace Elem Res, 1997, 56: 117~124
[6] Zhao L ,Cox AG,Ruzicka JA,et al.Molecular modeling and invitro activity of an HIV-I-encoded glutathione peroxidase.Proc Natl Acad Sci USA, 2000,97: 6356~6361
[7] King JC,Effects of reproduction on the bioavailability of calcium zinc and selenium.J Nutr, 2001,131(Suppl.4): 1355~1358
[8] Barrington JW,Taylor M,Smith S,et al.Selenium and recurrent miscarriage.J Obstet Gynaecol. 1997, 17: 199~200
[9] Scott R,Macpherson A.Selenium supplementation in sub-fertile human males.Br J Urol.1998,82: 76~80
[10] Finley JW,Penland JG . Adequacy or deprivation of dietary selenium in healthy men:clinical and psychological findings.J Trace Elem Exp Med, 1998, 11: 11~27
[11] Benton D,Cook R.Selenium supplementation improves mood in a double-blind crossover trial.Biol Psychiatry, 1991, 29: 1092~1098
[12] Olivieri O,Girelli D.Azzini M,et al.Low selenium status in the elderly influences thyroid hormones.Clin Sci, 1995, 89: 637~642.
[13] Miller S,Walker SW,Arthur JR,et al.Selenite protects human endothelial cells from oxidative damage and induces thioredoxin reductase.Clin Sci,2001, 100: 543~550
[14] Tanguy S,Boucher F,Besse S,et al.Trace elements and cardioprotection:increasing endogenous glutathione peroxidase activity by oral selenium supplementation in rats limits reperfusion-induced arrhythmias.J Trace Elem Med Biol, 1998, 12: 28~38
[15] Knekt P,Heliovaara M,Abo K,et al.Serum selenium serum alphatocopherol and the risk of rheumatoid arthritis.Epidemiology, 2000, 11: 402~405
[16] McCloy R.Chronic pancreatitis at Manchester.UK:focus on antioxidant therapy.Digestion, 1998, 59: 36~48
[17] Costello AJ.A randomized,controlled chemoprevention trial of selenium in familial prostate cancer:Rationale,recruitment,and design issues.Urology,2001,57: 182~184
[18] May SW, Pollock SH. Selenium-based antihypertensives. Rationale and potential. Drugs, 1998, 56(6)∶959
[19] Jacob C, Maret W, Vallee BL. Ebselen, a selenium-containing redox drug, releases zinc from metallothionein. Biochem Biophys Res Commun, 1998, 248(3)∶569
[20] Kondo H, Takahashi M, Niki E. Peroxynitrite-induced hemolysis of human erythrocytes and its inhibition by antioxidants. FEBS Lett, 1997, 413(2)∶236
[21] Engman L, Cotgreave I, Angulo M, et al. Diaryl chalcogenides as selective inhibitors of thioredoxin reductase and potential antitumor agents. Anticancer Res, 1997, 17(6D)∶4599
[22] Behne D,Kyriskcpoulos A,alcklosh M,et al.Two new selenproteins found in the prostatic glandular epithelium and the spermatid nuclei.Biomed Environ Sci,1997,10:340~345
[23]谢忠忱,王海宏.动物硒蛋白种类及生化功能的研究进展.中国实验动物学杂志, 2002, 12(3): 190~193
[24] Behne D, Hilmert H,Scheid S,et al. Evidence for specific selenium target tissues and new biologically important selenoproteins. Biochem Biophys Acta, 1988,966: 12~21
[25] Behne C,Kyriakopoeulos A, Weissnowak C,et al. Newly found selenium-containing proteins in the tissues of rats. Biol Tr Elem Res, 55: 99~110
[26] Burk TF, Hill KE. Regulation of selenoproteins.Ann Rev Nutr, 1993, 13: 65~81 [ 27 ] Hesketh JE.Tissues specific regulation of selenoenzyme gene expression duringselenium deficiency in rats.Biochem J, 1995, 311: 425~430
[28] Lei XG,Evenson JK,Rhompson KM,et al. Glutathione peroxidase and phGpx are differently regulated in rats by dietary selenium.J Nutr, 1995, 125: 1438~1446
[29] Burkman TP,Sutphin MS. Comparison of the effect of dietary selenium on selenoprotein expression in rat brain and liver.Biochem Acta, 1993, 1163: 176~184
[30] Arthur JR,Beckett FJ.New metabolic roles for selenium. Proc Nutr Soc, 1994, 53: 615~624
[31] Arthur JR. Non-glutathione peroxidase functions of selenium.Biotechnology in the food and feed:Proceedings of Alltech’s thirteenth annual symposium. 1997: 143~154
[32] Rotruck JT,Pope AL,Garther HE,et al.Selenium:biochem role as a component of glutathione peroxidase.Science, 1973,179: 588~590
[33] Hoekstra WG. Biochemical function of selenium and its relation to vitamin E.Fed Proc, 1975, 34: 2083~2089
[34] Arthur JR,Morrice PC,Nicol F,et al.The effect of selenium and copper deficiencies on glutathione S-transferase and glutathione peroxidase in rat liver.Biochem J, 1997, 248: 539~544
[35] Christensen MJ,Cammack PM,Wray CD.Tissue specificity of selenoprotein gene expression in rats.J Nutr Biochem, 1993, 6: 367~372
[36] Vadhanavikit S, Ganther HE. Selenium requirements of rats for normal hepatic and thyroidal 5’-deiodinase(Type I) activities.J Nutr, 1993, 123: 1124~1128
[37] Weitel F,Ursini F,Wendel A. Phospholipid hydroperoxide glutathione peroxidase in various mode organs during selenium deficiency and repletion .Biochem Biophys Acta, 1990, 1036: 88~94
[38] Yang J G,Hill KE,Burk RF. Dietary selenium intake controls rat plasma seleprotein peoncentration .J Nutr, 1989, 119: 1010~1012 [ 39 ] Geiger PG,Lin F,Girotti AW.Selenoperoxidase-mediated cytopotection against the damaging defects of terbuty hydroperoxide on leukemia cells.Free Radical Biol Med, 1993, 14: 251~266
[40] Kelner MJ,Bagnell RD,Uglik SF,et al. Heterologous expression of selenium-dependentglutathione peroxidase affords cellular resistance to paraquat.Arch Biochem Biophys, 1995, 323:40~46
[41] Cheng WH,Ho YS,Ross DA,et al. Over expression of cellular glutathione peroxidase does not affect expression of plasma glutathione peroxidase or phospholipids hydroperoxide glutathione peroxidase in mice offered diet adequate of deficient in selenium .J Nutr, 1997a, 127:675~680
[42] Cheng WH,Ho YS,Ross DA,et al. Cellular glutathione peroxidase knockout mice express normal levels of selenium-dependent plasma and phospholipids hydroperoxide glutathione peroxides in various tissues.J Nutr, 1997b, 127: 1445~1450
[43] Arthur JR,Nicol F and Beckett GJ. Hepatic iodothyronine deiodinase:The role of selenium.Biochem J, 1990a, 272: 537~540
[44] Arthur JR,Nicol J,Rae PWH,et al.Effect of selenium deficiency on the thyroid gland and on plasma and pituitary thyrotrophin and growth hormone concentrations in the rat.Clin Chem Enzyme Commun, 1990b, 3: 209~214 [ 45 ] Alavatore D,Bartha T,Harney JW,et al. Molecular biological and biochemical characterization of the human types 2 selenodeiodinase.Endocrinology, 1996, 137: 3308~3315
[46] Larsen PR ,Berry MJ. Nutritional and hormonal regulation of thyroid hormone deiodinases.Ann Rev Nutr, 1995, 15:323~352
[47] Beckett GJ,Beddows SE,Morrice PC,et al. Inhibition of hepatic deiodination of thyroxine caused by selenium deficiency in rats.Biochem J, 1987, 248: 443~447
[48] Beckett GJ,Macdougall DA,Nicol F,et al. Inhibition of type I and type II iodothyonine deiodinase activity in rat liver,kidney and brain produced by selenium deficiency. Biochem J, 1989, 259: 887~892
[49]呙于明.硒缺乏降低大鼠组织中5’-脱碘酶活性.营养学报.1992a,14:350~354
[50] Mitchell JH,Nicol F,Beckett GJ,et al. Selenoenzyme expression in thyroid and liver of second generation:selenium and iodine deficient rats.J Mol Endocrinol, 1996, 16: 259~267
[51] Herrmann JC. The properties of rat serum protein labeled the injection of sodiumselenite.Biochem.Biophys Acta, 1977, 500: 61~70
[52] Motsenbocher MA and Tappel AL. Aselenocysteine-containing selenium-transport protein in rat plasma.Biochem Biophys Acta, 1982, 719: 147~153
[53]杨晓光,田园,Hill KE,等.大鼠脑中血浆硒蛋白P.营养学报. 1997, 19: 375~378
[54]杨晓光,田园,Hill KE,等.大鼠脑中血浆硒蛋白P cDNA克隆.营养学报. 1998, 20: 1~7
[55] Burk RF,Hill KE,Awad JA,et al. Pathogenesis of diquat-induced liver necrosis in selenium deficient rats:Assessment of role of lipid peroxidation and selenoprotein P.Hepatology, 1995, 21: 561~569
[56] Chen WH,Ho HS,Valentine BA. Cellular glutathione peroxidase is the mediator of body selenium to protect against lethality in transgenic mice.J Nutr, 1998, 128: 1070~1076
[57] Vendeland SC,Beilstein MA,Chen CL,et al. Purification and properties of selenoprotein W from rat muscle.J Biological Chem, 1993, 268: 17103~17107 [ 58 ] Yeh JY,Beilstein MA,Forsberg NE,et al. Selenium influences tissues levels of selenoprotein W in sheep.J Nutr, 1997a, 127: 394~402
[59] Yeh JY,Beilstein MA,Gv QP,et al. Dietary selenium increase selenoprotein Levels in rat tissues.J Nutr, 1997b,127: 2165~2172
[60] Catadol L,Baig K,Oko R,et al. Developmental expression intracellular localization and selenium content of the cysteine-rich protein associated with the mitochondria capsules of mouse sperm.Mol Reprod Dev, 1996, 45: 320~331
[61] Sergey V,Novoselov,Mahadev Rao,Natalia V.Onoshko,et al.Selenoproteins and selenocysteine insertion system in the model plant cell system,Chlamydomonas reinhardtii[J] .The EMBO Journal,2002(21):3681-3693
[62] Atkins JF,Gsteland RF.The twenty-first amino acid.Nature,2000,407(6803):463-465
[63]蒋志华,牟颖,李维佳,阎岗林,罗贵民.生物合成硒蛋白机制的研究进展.生物化学与生物物理学报, 2002,34(4):395-399
[64] Stadtman TC.Selenocysteine.Annu Rev Biochem, 1996,65:83-100
[65] Zinoni F,Heider J,Bock A.Features of the formate dehydrogenase mRNA necessary for decoding of the UGA condon as selenocysteine.Proc Natl Acad SciUSA,1990,87(12):4660-4664
[66] Berry MJ,Banu I,Chen YY,Mandel SJ,Kieffer JD,Harney JW,Larsen PR.Recognition of UGA as a selenocysteine codon in type I deiodinase requires sequences in the 3’untranslated region.Nature,1991,353(6341):273-276
[67] Wilting R,Schorling S,Persson B C,Bock A.Selenoprotein synthesis in archaea:Identification of an mRNA element of Methanococcus jannaschii probably directing selenocysteine insertion. J Mol Biol, 1997, 266(4): 637~641
[68] Forchhammer K,Bock A.Selenocysteine synthase from Escherichia cloi.analysis of the reaction sequence.J Biol Chem. 1991, 266(10): 6324~6328 [ 69 ] Kromayer M,Wilting R,Tormay P,Bock A.Domain structure of the prokaryotic selenocystein specific elongation factor SelB.J Mol Biol, 1996, 262(4): 413~420
[70] Copeland PR,Fletcher JE,Carlson BA,Hatfield DL,Driscoll DM.A novel RNA binding protein SBP2,is required for the translation of mammalian selenoprotein mRNAs.EMBO J.2000, 19(2): 306~314
[71] Tujebajeva RM,Copeland PR,Xu XM,Carlson BA,Harney JW,Driscoll DM,Hatfield DI,et al.Decoding apparatus for eukaryotic selenocysteine insertion,EMBO Rep,2000,1(2): 158~163 [ 72 ] Liu Z,Reches M,Groisman I,Engelberg Kulka H.The nature of the minimal“selenocysteine insertion sequence”(SECIS) in Escherichia cloi .Nucleic Acids Res,1998,26(4): 896~902
[73] Chen GF,Fang I,Inouye M,Effect of the relative position of the UGA codon to the unique secondary structure in the fdhF Mrna on its decoding by selenocysteinyl tRNA in Escherichia cloi.J Boil Chem, 1993, 268(31): 23128~23131
[74] Baron C,Heider J,Bock A.Interaction of translation factor SELB with the formate dehydrogenase H selenopolypeptide mRNA .Proc Natl Acad Sci USA, 1993, 90(9): 4181~4185
[75] Li C,Reches M,Engelberg-Kulka H.The bulged nucleotide in the Escherichia cloi minimal selenocysteine participates in interaction with SelB:A genetic approach.J Bacteriol , 2000, 182(22): 6302~6307
[ 76 ] Liu Z,Reches M,Engelberg-Kulka H.A sequence in the Escherichia cloi fdhF“selenocysteine insertion Sequence”(SECIS) operates in the absence of selenium.J Mol Biol, 1999, 294(5): 1073~1086
[77] Poole ES,Brown CW, The identity of the base following the stop codon determines the efficiency of in vivo translational termination in Escherichia clo.EMBO J. 1995, 14(1): 151~158
[78] Sandman KE,Noren CJ,The efficiency of Escherichia cloi selenocysteine insertion is influenced by the immediate downstream nucleotide.Nucleic Acids Res, 2000,28(3): 755~761
[79] Hill KE,Lloyd RS,Burk RF.Conserval nucleotide sequences in the open reading frame and 3’untranslated region of selenoprotein P mRNA .Proc Natl Acad Sci USA. 1993,90(2): 537~541
[80] Berry MJ,Banu L,Harney JW,Larsen PR.Functional characterization of the eukaryotic SECIS elements which dired selenocysteine insertion at UGA codons.EMBO J, 1993,12(8):3315~3322
[81] Martin GW III,Harney JW,Berry MJ.Selenocysteine incorporation in eukaryotes:Insights into mechanism and efficiency from sequence,structure,and spacing proximity studies of the type I deiodinase SECIS element. RNA , 1996,2(2):171~182
[82] Fagegaltier D,Lescure A,Walczak R,Carbon P,Krol A.Structural analysis of new local features in SECIS RNA hairpins.Nucleic Acids Res, 2000,28(14):2679~2689
[83] Grundner-Culemann E,Martin GW III,Harney JW,Berry MJ.Two distinct SECIS structures capable of directing selenocysteine incorporation in eukaryotes.RNA, 1999,5(5):625~635 [ 84 ] Buettner C,Harney JW,Berry MJ.The Caenorhabditis elegans homologue of thioredoxin reductase contains a selenocysteine insertion sequence (SECIS) element that differs from mammalian SECIS elements but direct selenocysteine incorporation.J Boil Chem , 1999,274(31):21598~21602
[85] Kollmus H,Folhe L,McCarthy JE.Analysis of eukaryotic mRNA structures directing cotranslational incorporation of selenocysteine.Nucleic Acids Res, 1996,24(7):1195~1201
[86] Kryukov GV,Kryukov VM,Gladyshev VN.New mammalian searches for selenocysteine insertion sequence elements.J Biol Chem,1999,274(48): 33888~33897
[87] Ren X,Gao S,You D,Huang H,Liu Z,Mu Y,Liu J,et al.Cloning and expression of a single chain catalytic antibody that acts as a glutathione peroxidase mimic with high catalytic efficiency.Biochem J, 2001,359(2):369~374
[88] Tormay P,Bock A.Barriers to heterologous expression of a selenoprotein gene in bacteria.J Bacteriol, 1997,179(3): 576~582
[89]刘玉翠,陈英珠,辛春艳等.大豆硒蛋白药理作用研究.河北省科学院学报, 2000,17(3): 168~171
[90]向天勇,吴永尧,陈建英.天然大豆硒蛋白抗肿瘤作用研究,氨基酸和生物资源,2005,27(1): 10~12
[91]王卫真,唐家骏,彭安.富硒大蒜含硒蛋白的分离、鉴定和生物活性研究.生物化学杂志, 1989,5(3): 229~234
[92]杜明,赵镭,陈芳等.富硒灵芝中高抗氧化活力、高硒含量的水溶性硒蛋白的纯化.食品与发酵工业, 2006,32(2): 108~112
[93]杜明,赵镭,李朝睿等,富硒灵芝中一种新含硒蛋白的纯化、性质及其自由基清除活性研究.高等学校化学学报.2007,1:75~78
[94]王莲芳,窦春霞,张连富等,富硒食用菌中硒蛋白提取工艺研究,食品与发酵工业2007,33(1): 122~126
[95]宋家永,贾宏,吴雪松等.烟草硒蛋白研究进展.中国农学通报, 2007,9(23):159~161
[96] Terry N, Zayed AM, de Souza MP and Tarun AS. Selenium in Higher Plants. Annu. Rev. Plant Physiol. Plant Mol. Biol. 2000,51:401–32
[97] Sergey V.Novoselov,Mahadev Rao,Natalia V.Onoshko,et al.Selenoproteins and selenocysteine insertion system in the model plant cell system,Chlamydomonas reinhardtii[J] .The EMBO Journal,2002(21):3681-3693
[98] Gregory V.Kryukov,Sergi Castellano,Sergey V.Novoselov,et al.Characterizationg of Mammalian Selenoproteomes [J] .SCIENCE,2003(30):1439-1443
[99]刘琼,姜亮,徐辉碧等.疟蚊基因组中新硒蛋白的计算机识别[J].中国科学(C辑:生命科学), 2006,(05):451-458
[100]刘琼,陈平,徐辉碧等.生物体内硒蛋白的计算机识别方法[J].计算机与应用化学, 2006,(08):715-719
[101]张弛,吴永尧,彭振坤.植物硒的研究进展[J] .湖北民族学院学报(自然科学版),2002(20):58-62
[102]王翼飞,史定华.生物信息学-智能化算法及其应用.北京:化学工业出版社,2006
[103]楼士林.基因工程.北京:科学出版社,2002
[104]赵国屏.生物信息学.北京:科学出版社,2002
[105]茆诗松.贝叶斯统计.北京:中国统计出版社,1999
[106]史忠植.知识发现.北京:清华大学出版社,2002
[107] James O Berger.统计决策论及贝叶斯分析.贾乃光译.北京:中国统计出版社,1998
[108]李伍举.基因表达谱的生物信息学.军事医学科学院院刊,2001(1)
[109]边肇中,张学工.模式识别.第二版.北京:清华大学出版社,2000
[110] Gomord, V. and Faye, L. Posttranslational modification of therapeutic proteins in plants. Curr. Opin. Plant Biol, 2004, 7: 171~182
[111] Daniell, H. Molecular strategies for gene containment in transgenic crops. Nat. Biotechnol, 2002, 20: 581~586
[112]潘继承,汪劲松.植物基因工程中常用选择标记基因.基因工程,25~26
[113]陆晓春,郭长英,胡守荣.转基因植物中的标记基因.东北农业大学学报,2000,31(1):86~90
[114]段发平,梁承邺,黎垣庆.Bar基因和转Bar基因作物的研究进展.广西植物,2001,21(2):166~17
[115] Dale, P. J. The impact of hybrids between genetically modified crop plants and their related species: general considerations. Mol Ecol, 1994, 3:15~21
[116]刘谦,朱鑫泉.生物安全.北京:科学出版社,2000
[117]王忠华,夏英武.转基因植物中报告基因gus的表达及其安全性评价.生命科学,2000,12(5):207~210
[118] Hoffmann, A., Thimm, T. and Tebbe, C. C. Fate of plasmid-bearing, luciferase markergene tagged bacteria after feeding to the soil microarthropod Onychiurus fimatus (Collembola). FEMS Microbiol. Ecol, 1999, 30: 125~135
[119] Smalla, K., Gebhard, F. Van., Elsas. J. D., et al. Prevalence of nptII and Tn5 in kanamycin resistant bacteria from different environments. FEMS Microbial. Ecol, 1993, 13: 47~58
[120]赵和,吴志明,王海波等.抗除草剂转基因小麦生态安全评估(一)--禾本科麦田杂草、牧草禾主要近缘野生种的分布特点及生物学特性.河北农业科学,2000,(1):6~11
[121]赵和,曹建如,王海波等.抗除草剂转基因小麦生态安全评估(二)--普通小麦与其近缘野生属间和种间杂交研究.河北农业科学,2000,(3):6~9
[122] Burke, T., Seidler, R. and Smith, H. Ecological implication of transgenic plant release. Mol Ecol, 1994, 3(1): 1~89
[123]莫才清.标记基因在植物病害和流行规律研究中的应用.植物保护,1997(4):33~35
[124]王兴春,杨长登.转基因植物中的标记基因及其消除.中国生物工程杂志,2003, 23(4):19~22
[125] Finnegan, J. and McElroy, D. Transgene inactivation: plants fight back! Bio/Technology, 1994, 12: 883~888
[126] Xiang, D. F., Le, T. D., Strfanina, F., et al. Linear transgene constructs lacking vector backbone sequences generate low-copy-number transgenic plants with simple integration patterns. Transgenic Res, 2000, 9: 11~19
[127] Romano, A., Raemakers, K., Bernardi, J., et al. Transgenic organisation in potato after particle bombardment-mediated (co-) transformation using plasmids and gene cassettes. Transgenic Res, 2003, 12: 461~473
[128] Corneille, S., Lutz, K., Svab, Z., et al. Efficient elimination of selectable marker genes from the plastid genome by the CRE-lox site-specific recombination system. Plan J, 2001, 27: 171~178
[129] Klaus, S. M. J., Huang, F. C., Golds, T. J., et al. Generation of marker-free plastid transformants using a transiently cointegrated selection gene. Nat. Biotechnol, 2004, 22: 225~229
[130] Love, D. R., Pichler, F. B., Dodd, A., et al. Technology for high throughput screens: the present and future using zebrafish. Curr. Opin. Plant Biol, 2004, 15: 564~572
[131] Williams, G. P. Advances in high throughput screening. Drug Discov Today, 2004, 9: 515~517
[132] Germer, S., Holland, M. J. and Higuchi, R. High-throughput SNP allele frequency determination in pooled DNA samples by kinetic PCR. Genome Res, 2000, 10: 258~266
[133] Miller, C. J. and Attwood, T. K. Bioinformatics goes back to the future. Nat. Rev. Mol. Cell Biol, 2003, 4: 157~162
[134] Du, D. Z. and Hwang, F. K. Combinatorial Group Testing, 2nd Edition. World Scientific, Singapore, 2000
[135] Simon, R., Radmacher, M. D. and Dobbin, K. Design of studies using DNA micro- arrays genetic. Epidemiology, 2002, 23: 21~36
[136] Zou, G. H. and Zhao, H. Y. The impacts of errors in individual genotyping and DNA pooling on association studies. Genet. Epidemiol, 2004, 26: 1~10
[137] Mohlke, K. L., Erdos, M. L., Scott, L. J., et al. High-throughput screening for evidence of association by using mass spectrometry genotyping on DNA pools. Proc. Natl. Acad. Sci.U. S. A, 2002, 99: 16928~16933
[138] Harper, G., Pickett, S. D. and Green, D. V. S. Design of a compound screening collection for use in high throughput screening. Comb. Chem. High. Throughput. Screen, 2004, 7: 63~71
[139] Chen, J., Germer, S., Higuchi, R., et al. Kinetic polymerase chain reaction on pooled DNA: a high-throughput, high-efficiency alternative in genetic epidemiological studies. Cancer Epidemiol. Biomarkers Prev, 2002, 11: 131~136
[140] Dorfman, R. The detection of defective numbers of large population. Ann. Math. Statist, 1943, 14: 436~440
[141] Chen, C. and Swallow, W. Using group testing to estimate a proportion and to test the binomial model. Biometrics, 1990, 46: 1035~1046
[142] Hung, M. and Swallow, W. Robustness of group testing in the estimation of proportions. Biometrics, 1999, 55: 231~237
[143] Tebbs, J. and Swallow, W. Estimating ordered binomial proportions with the use of group testing. Biometrika, 2003, 90: 471~477
[144] Thomas, H. C., Charles, E. L., Ronald, L. R., et al. Introduction to Algorithms (MIT Electrical Engineering and Computer Science). The MIT Press, Five Cambridge Centre Cambridge, MA, USA, 1990
[145] Lin, X. R. and Pei, X. C. Basic Programming. NDI Press, Beijing, China, 1992