高压静电场处理花粉对白蜡属种间杂交的影响
|
摘要
本论文利用高压静电场处理白蜡属树种的花粉，通过提高花粉生命力促进白蜡属种间杂交亲和性和杂种生活力。为了揭示其分子机理，以经高压静电场处理和对照的水曲柳花粉为研究对象，利用mRNA差异显示(DDRT-PCR)和Reverse Northern杂交技术获得差异表达片段，并对基因片段进行测序和序列分析。所得结果如下：
1、白蜡属树种花粉萌发的比较适合培养基为：蔗糖10g·L~(-1)+琼脂1g·L~(-1)+尿素0.1g·L~(-1)+硼酸1g·L(-1)+硝酸钾0.1g·L~(-1)+硫酸镁0.2g·L~(-1)+硝酸钙0.1g·L~(-1)+硫酸锌0.1mg·L~(-1)+钼酸钠20 mg·L~(-1)+NAA10mg·L~(-1)+6-BA1mg·L~(-1)，pH6.5。
2、本试验测定株间花粉生活力最高相差30.9％，说明白蜡属株间花粉生命力差异明显。
3、将花粉4℃密封保存有利于保持花粉的生命力。
4、经适宜的高压静电场处理，水曲柳的花粉萌发率可比对照高出19.7％。
5、经适宜场强处理的白蜡属花粉与水曲柳雌株杂交，座果率明显提高，最多提高13.5％。
6、大叶白蜡、小叶白蜡和绒毛白蜡花粉经高压静电场处理后杂交得到的种子在种长、种宽、千粒重三方面几乎都高于未处理得到的杂交种子。种长差别最大的品种前者比后者高出0.25cm(9.40％)；种宽差别最大的品种前者比后者高出0.06cm(9.84％)；千粒重差别最大的品种前者比后者高出13.71g(25.90％)。
7、经高压静电场处理花粉后得到的杂交苗在苗高、地茎、光和速率、叶绿素含量方面都明显高于对照苗。
8、改良的CTAB法是提取水曲柳花粉总RNA的有效方法。
9、水曲柳花粉经高压静电场处理后有特异基因表达。通过Reverse Northern斑点杂交的检测鉴定，共获得6个差异基因片段。通过序列测定及分析，发现6个基因片段分别与拟南芥的核苷控制的离子通道基因、番茄叶绿体的铜锌超氧化物歧化酶(SOD)基因、番茄叶绿体的铜锌超氧化物歧化酶(SOD)基因、拟南芥的核酸调控的离子通道蛋白、与番茄的激素诱导的蛋白酶抑制剂基因、苜蓿科细胞色素P450单氧酶有着较高同源性。氨基酸序列同源性分别高达98％、95％、96％、99％、93％、93％。
In this paper the pollens of the species of Fraxinus Linn were treated with high voltage static, which promoted the cross-compatibility by enhancing the vitality of their pollen. In order to study the molecule mechanism of it, both of the control and pollens dealt with high voltage static field were taken for RNA extract.The DNA fragments of differential expressed genes were gotten by DDRT-PCR and were confirmed by Reverse Northern dot blotting, and then they were sequenced and analyzed. The main results were as follows:
1, The suitable culture medium for the pollens germination of Fraxinus Linn is : sugar 10 g.L~(-1)+agar 1 g·L~(-1)+urea 0.1 g·L~(-1)+boric acid 0.5 g·L~(-1)+nitre 0.1 g·L~(-1)+bitter salt 0.2 g·L~(-1) +calcium nitrate 0.1 g·L~(-1)+zinc salfate 0.1 mg·L~(-1)+sodium molybdate 20 mg·L~(-1)+NAA10 mg·L~(-1)+6-BA 1mg·L~(-1),pH 6.5.
2、The differences of pollens vitality between individual plants were significant, which up to 30.9 percent at the most among the trees experimented in this paper.
3、To keep higher vitality ,pollens should be sealed after dry and stored below 4℃in refrigerator.
4、The germination rate of pollen of Fraxinus mandshurica treated with the proper high voltage static field was 19.7 percent higher than that of control.
5、The pollens treated with proper high voltage static field could promote hybrid between the species of Fraxinus Linn and get a higher fructification rate, 13.5 percents higher than control at the most.
6、The hybrid seeds of Fraxinus sogdiana Bunge, Fraxinus velutina Torr. and Fraxinus rhynchophylla obtained by pollen processed by proper high voltage static shared higher data in seed length, seed width and kilo granule weight than control, and the higher values of these were0.25centimeters(9.40percents),0.06centimeters(9.84percents)and13.71gram (25.9percents) at the most.
7、F1 seedlings from pollen treated by high voltage static were notably different from control(not treatment)in seedling height, ground stem width, photosynthesis rate and chlorophyll content.
8、Improved CTAB method is effective in extracting the RNA in the pollen of Fraxinus mandshurica.
9、Genes in the pollen of Fraxinus mandshurica expressed differential after processed by high voltage static field. Identified by reverse northern dot blotting, 6 differential expressed gene fragments were gotten. By sequencing and analyzing, we found that the fragments have great identity with the gene for cyclic nucleotide-gated cation channel in Arabidopsis thaliana, with the gene for calmodulin binding / cyclic nucleotide binding / ion channel in Arabidopsis thaliana, with the gene for chloroplast Cu-Zn superoxide dismutase in tomato,with the gene for putative cyclic nucleotide-regulated ion channel protein in Arabidopsis thaIiana, with the gene for auxin-induced proteinase inhibitor (ARPI) gene in Lycopersicon esculentum, and with the cytochrome P450 monooxygenase CYP76E1 (CYP76E1) gene in Medicago truncatula respectively.There nucleotide homologous were 98percents, 95percents, 96percents, 99percents, 93percents and 93percents.
1、白蜡属树种花粉萌发的比较适合培养基为：蔗糖10g·L~(-1)+琼脂1g·L~(-1)+尿素0.1g·L~(-1)+硼酸1g·L(-1)+硝酸钾0.1g·L~(-1)+硫酸镁0.2g·L~(-1)+硝酸钙0.1g·L~(-1)+硫酸锌0.1mg·L~(-1)+钼酸钠20 mg·L~(-1)+NAA10mg·L~(-1)+6-BA1mg·L~(-1)，pH6.5。
2、本试验测定株间花粉生活力最高相差30.9％，说明白蜡属株间花粉生命力差异明显。
3、将花粉4℃密封保存有利于保持花粉的生命力。
4、经适宜的高压静电场处理，水曲柳的花粉萌发率可比对照高出19.7％。
5、经适宜场强处理的白蜡属花粉与水曲柳雌株杂交，座果率明显提高，最多提高13.5％。
6、大叶白蜡、小叶白蜡和绒毛白蜡花粉经高压静电场处理后杂交得到的种子在种长、种宽、千粒重三方面几乎都高于未处理得到的杂交种子。种长差别最大的品种前者比后者高出0.25cm(9.40％)；种宽差别最大的品种前者比后者高出0.06cm(9.84％)；千粒重差别最大的品种前者比后者高出13.71g(25.90％)。
7、经高压静电场处理花粉后得到的杂交苗在苗高、地茎、光和速率、叶绿素含量方面都明显高于对照苗。
8、改良的CTAB法是提取水曲柳花粉总RNA的有效方法。
9、水曲柳花粉经高压静电场处理后有特异基因表达。通过Reverse Northern斑点杂交的检测鉴定，共获得6个差异基因片段。通过序列测定及分析，发现6个基因片段分别与拟南芥的核苷控制的离子通道基因、番茄叶绿体的铜锌超氧化物歧化酶(SOD)基因、番茄叶绿体的铜锌超氧化物歧化酶(SOD)基因、拟南芥的核酸调控的离子通道蛋白、与番茄的激素诱导的蛋白酶抑制剂基因、苜蓿科细胞色素P450单氧酶有着较高同源性。氨基酸序列同源性分别高达98％、95％、96％、99％、93％、93％。
In this paper the pollens of the species of Fraxinus Linn were treated with high voltage static, which promoted the cross-compatibility by enhancing the vitality of their pollen. In order to study the molecule mechanism of it, both of the control and pollens dealt with high voltage static field were taken for RNA extract.The DNA fragments of differential expressed genes were gotten by DDRT-PCR and were confirmed by Reverse Northern dot blotting, and then they were sequenced and analyzed. The main results were as follows:
1, The suitable culture medium for the pollens germination of Fraxinus Linn is : sugar 10 g.L~(-1)+agar 1 g·L~(-1)+urea 0.1 g·L~(-1)+boric acid 0.5 g·L~(-1)+nitre 0.1 g·L~(-1)+bitter salt 0.2 g·L~(-1) +calcium nitrate 0.1 g·L~(-1)+zinc salfate 0.1 mg·L~(-1)+sodium molybdate 20 mg·L~(-1)+NAA10 mg·L~(-1)+6-BA 1mg·L~(-1),pH 6.5.
2、The differences of pollens vitality between individual plants were significant, which up to 30.9 percent at the most among the trees experimented in this paper.
3、To keep higher vitality ,pollens should be sealed after dry and stored below 4℃in refrigerator.
4、The germination rate of pollen of Fraxinus mandshurica treated with the proper high voltage static field was 19.7 percent higher than that of control.
5、The pollens treated with proper high voltage static field could promote hybrid between the species of Fraxinus Linn and get a higher fructification rate, 13.5 percents higher than control at the most.
6、The hybrid seeds of Fraxinus sogdiana Bunge, Fraxinus velutina Torr. and Fraxinus rhynchophylla obtained by pollen processed by proper high voltage static shared higher data in seed length, seed width and kilo granule weight than control, and the higher values of these were0.25centimeters(9.40percents),0.06centimeters(9.84percents)and13.71gram (25.9percents) at the most.
7、F1 seedlings from pollen treated by high voltage static were notably different from control(not treatment)in seedling height, ground stem width, photosynthesis rate and chlorophyll content.
8、Improved CTAB method is effective in extracting the RNA in the pollen of Fraxinus mandshurica.
9、Genes in the pollen of Fraxinus mandshurica expressed differential after processed by high voltage static field. Identified by reverse northern dot blotting, 6 differential expressed gene fragments were gotten. By sequencing and analyzing, we found that the fragments have great identity with the gene for cyclic nucleotide-gated cation channel in Arabidopsis thaliana, with the gene for calmodulin binding / cyclic nucleotide binding / ion channel in Arabidopsis thaliana, with the gene for chloroplast Cu-Zn superoxide dismutase in tomato,with the gene for putative cyclic nucleotide-regulated ion channel protein in Arabidopsis thaIiana, with the gene for auxin-induced proteinase inhibitor (ARPI) gene in Lycopersicon esculentum, and with the cytochrome P450 monooxygenase CYP76E1 (CYP76E1) gene in Medicago truncatula respectively.There nucleotide homologous were 98percents, 95percents, 96percents, 99percents, 93percents and 93percents.
引文
[1] 梁运章，于永方．静电研究与进展[M]．呼和浩特：内蒙古大学出版社，1992
[2] 王秀文，王荣毅．静电高压场中植物生长的研究[A]．中国物理学会静电专业委员会学术及交流资料[C]．北京：中国物理学会，1983，21-25．
[3] 叶家明．静电高压场对植物有丝分裂的影响[A]．中国物理学会静电生物效应在农业上的应用学术交流资料[C]．北京：中国物理学会，1984，76-77．
[4] 肖艳辉，何金明．磁场对农作物的生物学效应[J]．韶关学院学报．2003，24(6)：45-47
[5] 林任荣．磁处理技术在农业中的应用[J]，福建农业大学学报，1999，28(4)：509-512．
[6] S.Pietruszewski.磁化处理种子对小麦产量的影响[J]．国外农学-麦类作物，1995(1)：1718，34
[7] 谢菊芳，汪爱珍，张菁等，静电场中植物细胞畸变与分析[J]．湖北大学学报，2000，22(3)：253-255
[8] 李怀智，庞金安．物理方法处理种子对黄瓜生长发育的影响研究进展[J]．农业与技术，2003，23(2)：47-49
[9] 张振球．静电生物效应[M]．北京：万国学术出版社，1989：1
[10] Frohlich H.What Are Non-Thermal Electric Biological Effects [J].Bioelectro magnetics,1982,13:45-46
[11] 高云飞，郑世民．高压静电场生物效应的产生机制[J]．畜牧兽医科技信息2004，(10)：17-18
[12] 胡玉才，袁泉，陈奎孚．农业生物的电磁环境效应研究综述[J]．农业工程学报，1999，15(2)：15-20
[13] Wulfsen,H.-P.Hauber, D. Schiemann,et al.Expression of mRNA for Voltage-Dependent and Inward-Rectifying K Channels in GH3/B6 Cells and Rat Pituitary[J].Journal of Neuroendocrinology, 2000, 12:263-272.
[14] David Glahn,Richard Nuccitelli.Voltage-clamp study of the activation currents and fast block to polyspermy in the egg of Xenopus laevis Develop[J].Growth Differ, 20133,(45):187-197.
[15] ChenLei,LiuChangjin,Tang Ming,et al.Action of aluminum on high voltage-dependent calcium current and its modulation by ginkgolide B[J].Acta Pharmacologica Sinica, 2005,26(5): 539-545.
[16] Claudia L, Trevin6,Ricardo Felix Castellano,et al.Expression and differential cell distribution of low-threshold Ca~(2+) channels in mammalian male germ cells and sperm[J].FEBS Letters,2004(563): 87-92.
[17] Andrea Marquart,Veit Flockerzi.al-βinteraction in voltage-gated cardiac L-type calcium channels[J].FEBS Letters,1997(407): 137-140.
[18] Myoung-Goo Kang,Ricardo Felix,Kevin P, et al.Campbell.Long-term regulation of voltage-gated Ca~(2+) channels by gabapentin[J].FEBS Letters,2002(528):177-182.
[19] Juan Manuel Arias,Janet Murbartian,Iuliia Vitko,et al.Transfer of b subunit regulation from high to low voltage-gated Ca~(2+) channels[J].FEBS Letters,2005 (579):3907-3912.
[20] Jan A. Gorter, Aren J. Borgdorff, Erwin A. van, Vliet,et al.Differential and long-lasting alterations of high-voltage activated calcium currents in CA1 and dentate granule neurons after status epilepticus[J].European Journal of Neuroscience, 2002(16): 701-712.
[21] Mark E Williams,Mark S Washburn,Michael Hans,et al.Structure and Functional Characterization of a Novel Human Low-Voltage Activated Calcium Channel[J]. Journal of Neurochemistry,1999(72):791 -799.
[22] Chen Chen,Xu Ruwei,Iain J Clarke,et al.Diverse intracellular signalling systems used by growth hormone releasing hormone in regulating voltage-gated Ca~(2+) or K~+ channels in pituitary somatotropes[J].Immunology and Cell Biology,2000(78):356-368.
[23] Toshio Ohta,Arun R.Wakade,Yoshikazu Nakazato,et al.Ca2~+ -dependent K~+ current and exocytosis in responses to caffeine and muscarine in voltage-clamped guinea-pig adrenal chromaffin cells[J]. Journal of Neurochemistry,2001 (78):1243 -1255.
[24] Hans-Georg Fischer, Claudia Eder.Voltage-gated k~+ currents of mouse dendritic cells [J]. FEBS Letters,1995(373):127-130.
[25] Luca Monticelli,Kindal M.Robertson,Justin L.MacCallum,et al.Computer simulation of the KvAP voltage-gated potassium channel: steered molecular dynamics of the voltage sensor[J].FEBS Letters,2004(564):325-332.
[26] Rita Casadio,Irene Jacoboni,Angela Messina,et al.A 3D model of the voltage-dependent anion channel (VDAC) [J].FEBS Letters,2002(520):1-7.
[27] Kropacheva T N,Raap J.Voltage-dependent interaction of the peptaibol antibiotic zervamicin Ⅱ with phospholipid vesicles[J].FEBS Letters,1999(460):500-504.
[28] Nitzsche W, Kung.Control of enzyme - distribution in enzyme -membrane by electric field[M]．Handbook of plant Cell Culture．北京：中国科技出版社，1994：728-805
[29] 陈怡平，王勋陵．He-Ne激光处理对菘蓝幼苗光合作用的影响[J]．激光技术，2004，28(1)：42-44
[30] 王振邦．高压静电场对绿豆种子活力影响的机理探讨[J]．陕西教育学院学报，2004，20(2)：116-117．
[31] 吴旭红，孙为民，张红燕．高压静电场对南瓜种子萌发及幼苗生长的生物学效应[J]．种子，2004，23(2)：67-68．
[32] 王清元，卢贵忠，赵玉清．高压静电场对水稻种子萌发的试验研究[J]．云南农业大学 学报，2005，20(1)：47-50．
[33] 岛山英雄．野外植物の生体电位[J]，静电气学会，1982，(5)：276-284
[34] 赵剑，杨文杰，马福荣等．高压静电场(HVEF)对苜蓿叶片愈伤组织诱导的影响[J]．生物物理学报，1996，(3)：517-520
[35] 赵剑，杨文杰，马福荣等．静电场对苜蓿愈伤组织抗寒能力的影响[J]．生物物理学报，2000，16(2)：406-411
[36] 叶家明．静电处理对细胞有丝分裂的影响[J]．东北师大学报(自然版)，1985，(1)：61-66
[37] 马福荣．静电场对植物光合器官结构和功能的影响[J]．生物物理学报，1994，10(3)：468-472
[38] 张振球．静电生物效应[M]．北京：万国学术出版社．1989：1-58
[39] L E Murr.Biophysics of plant growth in an electro static field[J].Nature,1965, (206): 467- 470
[40] G H Sidaway, G F A spray. Influence of electro static field on plant respiration[J]. International Journal of Biometeorogy, 1968, (12) : 321- 329
[41] A P Krueger. Biological impact of small air ions[J].Science,1976,19(3):1209- 1213
[42] 三浦丰彦．高压电场の长期暴露の影响[J]．劳动科学，1979，55(12)：682-688
[43] 伊板等．高电界の生体系の影响[J]．静电气学会志，1975，(9)：176
[44] 唯薇，丁晓东，霍俊伟．草原樱桃与欧洲甜樱桃远缘杂交不亲和原因初探[J]．东北农业大学学报30(2)：148-153
[45] 万贤国．电离辐射技术在作物远缘杂交上的应用[J]．湖南农业科学，1994(3)：45-47
[46] 李玉晖，陈学森，郑洲．果树远缘杂交育种研究进展[J]．山东农业人学学报(自然科学版)，2003，34(1)：139-143
[47] 工树彦．石锦凤．李莉等．禾本科小麦族植物远缘杂交的障碍及其克服[J]．中国草地，2003，25(2)：59-62
[48] 宋道军，陈若雷，尹若春等．离子束介导植物分子超远缘杂交研究[J]．自然科学进展，2001，11(3)：327-330
[49] 苏学合，高国强，时香玉等．利用花粉辐照技术进行棉花种间远缘杂交研究[J]．山东农业科学，2000(2)：8-10
[50] 张国庆，周伟军，姚先伶等．芸薹属植物远缘杂交研究现状[J]．山西农业科学2001，29(4)：25-30
[51] 刘琳莉，强胜，宋小玲等．用荧光显微镜技术观察药用野生稻(Oryza officinalis Wall)和转基因水稻的不亲和性[J]．中国农业科学2004，37(4)：469-472
[52] 邢少辰，周开达，蔡玉红，植物远缘杂交的研究[J]．吉林农业科学，2000，25(4)：18-21
[53] 高新起，王秀玲．植物远缘杂交的障碍及其克服[J]．生物学通报，1998，33(12)
[54] 王幼平，罗鹏，何兴金．诸葛菜与芸苔属属间杂交时花粉-雌蕊相互作用的研究[J]．广 西植物，1997，17(4)：371-374
[55] 向旭．影响荔枝花粉活力的化学因子研究[J]．广东科学农业．2000．(06)：56-60
[56] 郭光明，张福锁，尚忠林．硼对百合花粉萌发过程中细胞内游离钙离子的影响[J]．中国农业大学学报2002，7(5)：32-37
[57] 王玉芹，刘昌智，邓洪民．应用10B研究不同油菜品种花器官的硼素基因型差异[J]．中国油料，1993，(4)：11-14
[58] 沈康，沈振国，徐汉卿等．油菜硼素营养与结实性的研究[J]．作物学报，1993，19(6)：539-545
[59] 褚天铎，陈家驹，刘昌智等．油菜缺硼花而不实原因的探讨[J]．植物营养与肥料学报，1996，2(1)：23-31
[60] 任沪生，陈仲西，肖昌珍．油菜萎缩不实病防治研究．中国科学院微量元素学术交流会汇刊[M]．北京：科学出版社，1980：87-98
[61] 张秀省，沈振国，沈康．硼对油菜花器官发育和结实性的影响[J]．土壤学报，1994，31(2)：146-152
[62] 沈振国，张秀省，王振宇等．硼素营养对油菜花粉萌发的影响[J]．中国农业学，1994，27(1)：51-56
[63] 杨弘远．钙在有花植物受精过程中的作用[J]．植物学报1999，41(10)：1027-1035
[64] 唐前瑞，陈友云．连蕊茶花粉发芽适宜培养基的探讨[J]．湖南林业科技．2004．31．(1)：32-35
[65] 薛妙男，刘华英．沙田柚花粉低温贮藏的研究[J]．广西植物．2000．20(4)：367-370
[66] 张玉进，张兴国．魔芋花粉的低温和超低温保存[J]．园艺学报．2000．27(2)：139-140
[67] 周莉，代力民，徐杰．丁香花粉生命力及贮藏力的研究[J]．沈阳农业大学学报，2003-02．34(1)：16-19
[68] F.奥斯伯，R.布伦特，R.E.金斯顿等．精编分子生物学指南[M]．北京：科学出版社，1998：120-130
[69] 崔大祥，闰小君，苏成芝．差异表达基因克隆技术的进展[J]．生命的化学，1999(5)
[70] Bauer D,Muller H,Reich J.et al.Identification of differentially expressed mRNA species by an improved display technique(DDRT-PCR)[J].Nucleic Acids Res,1993, 21 (18):4272-4280
[71] Ito T, Kito K, Adati N,etal.Fluorescent differential display: Arbitraryly primed RT-PCR fingerprinting on an automated .DNA sequencer[J].FEBE Letters,1994,35(23):231-236
[72] Liang P ,Zhu WM,Zhang XY, et al.Differential display using one-base anchored oligo-dT primers[J].Nucleic Acids Res,1994,22(25):5763-5764
[73] 陈斌，袁建刚，潘美辉等．差异显示反转录PCR技术的建立和改进[J]．基础医学与临床，1996：63-67
[74] Callard D, Lescure B, Mazzolini L. A method for the elimination of false positives generated by them. RNA differential display technique[J]. Bio Techniques, 1994(16): 1096-1103
[75] Corton JC, Gustafsson JA. Increased efficiency in screening large numbers of cDNA fragments generated by differential display[J]. Biotechniques, 1997. 22(5): 802-804. 806. 808
[76] Doss RP. Differential display without radioactivity-A modified procedure[J]. Bio Techniques 1996, 21(3): 408-412
[77] Gottschlich S, Goeroegh T, Folz BJ, et al. Optimized differential display and reamplification parameters for silverstaining[J]. Res Comm Mol Path Pharm, 1997, 97 (2): 237-240
[78] Hadman M, Adam BL, Wright GL, et al. Modification to the differential display technique reduces background and increase sensitivity[J]. Anal Biochem, 1995, 226: 383-386
[79] Ito T, Kito K, AdatiN, et al. Fluorescent differential display: Arbitraryly primed RT-PCR fingerprinting on an automated DNA sequencer[J]. FEBE Letters, 1994, 35(23): 231-236
[80] Ito T, Sakaki Y. Fluorescent differential display method for high speed scanning of tissue-or cell-specific transcripts[J]. Metods Mol Genet. 1996, 8: 229-245
[81] Liang P, Averboukh L, Pardee AB. Distribution and cloning of eukaryotic mRNAs by means of differential display: Refinements and optimization[J]. Nucleic Acids Res, 1993, 21: 3269-3275
[82] Liang P, Pardee AB. Differential display methods and protocols (methods in Molecular Biology, Vol. 85)[M]. Totowa, VewJersey: HumanaPress, I997, 45-103
[83] Liang Peng, Arthur B Pardee. Differential display of eukaryotic messenger RNA by means of the polymerase chain reaction[J]. Science, 1992, 257(14): 967-971
[84] Liu C, Raghothame KG. Practical method for cloning cDNAs generated in an mRNA differential display[J]. Bio Techniques, 1996, 20(4): 576-580
[85] Lohmann J, Schickle H, Bosch T C G. REN display, a rapid and efficient method for noradioactive differential display and mRNA isolation[J]. BioTchniques, 1995, 18(2): 200-202
[86] Luce MJ. Minimizing false positives in differential display[J]. Bio Techniques 1998. 24 (5): 766-768
[87] Malhotra K, Foltz L, Mahoney WC, et al. Interaction and effect of annealing temperature on primers used in differential, display RT-PCR[J]. Nucleic Acids Res, 1998, 26 (3): 854-856
[88] Matz MV, Lukyanov SA. Different strategies of differential display: areas of application [J].Nucleic Acids Res,1998,6(24):5537-5543
[89] Miele G.Elimination of false positives generated through PCR-reamplification of differential display cDNA[J].Bio techniques,1998,25(1):138-144
[90] Mol L, Miller HM,Li J,et al. Improvements to the differential display method for gene analysis[J]. Bio Biophysical Res Communications, 1994,199(2):564-569
[91] Schneiderbauer A, Sandermann H Jr, Ernst D.Isolation of functional RNA from plant tissues rich in phenolic compounds[J]. Anal Biochern,1991,(197):91-95
[92] Lewinsohu E. Steele CL, Croteau R. Simple isolation of functional RNA from woody stems of gymnosperms[J]. Plant Mol Biol Rep,1994,(12):20-25
[93] 王立秋．激光诱变的生物学效应及其在动植物遗传育种上的应用[J]．激光生物学报，1997．6(2)：1097-1102
[94] 刘家克．中国蔬菜栽培学[M]．中国农业科学院蔬菜研究所主编．农业出版社．1988：48-49
[95] 王玉成，薄海侠，杨传平．胡杨、柽柳总RNA提取方法的建立[J]．东北林业大学学报，2003，31(5)：99-100
[96] 徐祖荫，孙启跃，李干洲．不同破壁法对花粉提取液中可溶性营养成分的影响[J]．蜜蜂杂志月刊，1994，(1)：6-10
[97] 姚成义，赵洁．钙和硼对蓝猪耳花粉萌发及花粉管生长的影响[J]．武汉植物学研究，2004，22(1)：1-7
[98] 唐维，张星海．花粉破壁方法的研究进展[J]．食品与发酵工业，29(2)：86-92
[99] Kong Hai-Yan, Jia Gui-Xia.Calcium Distribution During Pollen Development of Larixprincipis-rupprechtii[J],植物学报，2004，46(7):60-76
[100] 李安平，谢碧霞，常银子．马尾松花粉破壁技术及其发酵饮料[J]．东北林业大学学报，32(1)，11-13
[2] 王秀文，王荣毅．静电高压场中植物生长的研究[A]．中国物理学会静电专业委员会学术及交流资料[C]．北京：中国物理学会，1983，21-25．
[3] 叶家明．静电高压场对植物有丝分裂的影响[A]．中国物理学会静电生物效应在农业上的应用学术交流资料[C]．北京：中国物理学会，1984，76-77．
[4] 肖艳辉，何金明．磁场对农作物的生物学效应[J]．韶关学院学报．2003，24(6)：45-47
[5] 林任荣．磁处理技术在农业中的应用[J]，福建农业大学学报，1999，28(4)：509-512．
[6] S.Pietruszewski.磁化处理种子对小麦产量的影响[J]．国外农学-麦类作物，1995(1)：1718，34
[7] 谢菊芳，汪爱珍，张菁等，静电场中植物细胞畸变与分析[J]．湖北大学学报，2000，22(3)：253-255
[8] 李怀智，庞金安．物理方法处理种子对黄瓜生长发育的影响研究进展[J]．农业与技术，2003，23(2)：47-49
[9] 张振球．静电生物效应[M]．北京：万国学术出版社，1989：1
[10] Frohlich H.What Are Non-Thermal Electric Biological Effects [J].Bioelectro magnetics,1982,13:45-46
[11] 高云飞，郑世民．高压静电场生物效应的产生机制[J]．畜牧兽医科技信息2004，(10)：17-18
[12] 胡玉才，袁泉，陈奎孚．农业生物的电磁环境效应研究综述[J]．农业工程学报，1999，15(2)：15-20
[13] Wulfsen,H.-P.Hauber, D. Schiemann,et al.Expression of mRNA for Voltage-Dependent and Inward-Rectifying K Channels in GH3/B6 Cells and Rat Pituitary[J].Journal of Neuroendocrinology, 2000, 12:263-272.
[14] David Glahn,Richard Nuccitelli.Voltage-clamp study of the activation currents and fast block to polyspermy in the egg of Xenopus laevis Develop[J].Growth Differ, 20133,(45):187-197.
[15] ChenLei,LiuChangjin,Tang Ming,et al.Action of aluminum on high voltage-dependent calcium current and its modulation by ginkgolide B[J].Acta Pharmacologica Sinica, 2005,26(5): 539-545.
[16] Claudia L, Trevin6,Ricardo Felix Castellano,et al.Expression and differential cell distribution of low-threshold Ca~(2+) channels in mammalian male germ cells and sperm[J].FEBS Letters,2004(563): 87-92.
[17] Andrea Marquart,Veit Flockerzi.al-βinteraction in voltage-gated cardiac L-type calcium channels[J].FEBS Letters,1997(407): 137-140.
[18] Myoung-Goo Kang,Ricardo Felix,Kevin P, et al.Campbell.Long-term regulation of voltage-gated Ca~(2+) channels by gabapentin[J].FEBS Letters,2002(528):177-182.
[19] Juan Manuel Arias,Janet Murbartian,Iuliia Vitko,et al.Transfer of b subunit regulation from high to low voltage-gated Ca~(2+) channels[J].FEBS Letters,2005 (579):3907-3912.
[20] Jan A. Gorter, Aren J. Borgdorff, Erwin A. van, Vliet,et al.Differential and long-lasting alterations of high-voltage activated calcium currents in CA1 and dentate granule neurons after status epilepticus[J].European Journal of Neuroscience, 2002(16): 701-712.
[21] Mark E Williams,Mark S Washburn,Michael Hans,et al.Structure and Functional Characterization of a Novel Human Low-Voltage Activated Calcium Channel[J]. Journal of Neurochemistry,1999(72):791 -799.
[22] Chen Chen,Xu Ruwei,Iain J Clarke,et al.Diverse intracellular signalling systems used by growth hormone releasing hormone in regulating voltage-gated Ca~(2+) or K~+ channels in pituitary somatotropes[J].Immunology and Cell Biology,2000(78):356-368.
[23] Toshio Ohta,Arun R.Wakade,Yoshikazu Nakazato,et al.Ca2~+ -dependent K~+ current and exocytosis in responses to caffeine and muscarine in voltage-clamped guinea-pig adrenal chromaffin cells[J]. Journal of Neurochemistry,2001 (78):1243 -1255.
[24] Hans-Georg Fischer, Claudia Eder.Voltage-gated k~+ currents of mouse dendritic cells [J]. FEBS Letters,1995(373):127-130.
[25] Luca Monticelli,Kindal M.Robertson,Justin L.MacCallum,et al.Computer simulation of the KvAP voltage-gated potassium channel: steered molecular dynamics of the voltage sensor[J].FEBS Letters,2004(564):325-332.
[26] Rita Casadio,Irene Jacoboni,Angela Messina,et al.A 3D model of the voltage-dependent anion channel (VDAC) [J].FEBS Letters,2002(520):1-7.
[27] Kropacheva T N,Raap J.Voltage-dependent interaction of the peptaibol antibiotic zervamicin Ⅱ with phospholipid vesicles[J].FEBS Letters,1999(460):500-504.
[28] Nitzsche W, Kung.Control of enzyme - distribution in enzyme -membrane by electric field[M]．Handbook of plant Cell Culture．北京：中国科技出版社，1994：728-805
[29] 陈怡平，王勋陵．He-Ne激光处理对菘蓝幼苗光合作用的影响[J]．激光技术，2004，28(1)：42-44
[30] 王振邦．高压静电场对绿豆种子活力影响的机理探讨[J]．陕西教育学院学报，2004，20(2)：116-117．
[31] 吴旭红，孙为民，张红燕．高压静电场对南瓜种子萌发及幼苗生长的生物学效应[J]．种子，2004，23(2)：67-68．
[32] 王清元，卢贵忠，赵玉清．高压静电场对水稻种子萌发的试验研究[J]．云南农业大学 学报，2005，20(1)：47-50．
[33] 岛山英雄．野外植物の生体电位[J]，静电气学会，1982，(5)：276-284
[34] 赵剑，杨文杰，马福荣等．高压静电场(HVEF)对苜蓿叶片愈伤组织诱导的影响[J]．生物物理学报，1996，(3)：517-520
[35] 赵剑，杨文杰，马福荣等．静电场对苜蓿愈伤组织抗寒能力的影响[J]．生物物理学报，2000，16(2)：406-411
[36] 叶家明．静电处理对细胞有丝分裂的影响[J]．东北师大学报(自然版)，1985，(1)：61-66
[37] 马福荣．静电场对植物光合器官结构和功能的影响[J]．生物物理学报，1994，10(3)：468-472
[38] 张振球．静电生物效应[M]．北京：万国学术出版社．1989：1-58
[39] L E Murr.Biophysics of plant growth in an electro static field[J].Nature,1965, (206): 467- 470
[40] G H Sidaway, G F A spray. Influence of electro static field on plant respiration[J]. International Journal of Biometeorogy, 1968, (12) : 321- 329
[41] A P Krueger. Biological impact of small air ions[J].Science,1976,19(3):1209- 1213
[42] 三浦丰彦．高压电场の长期暴露の影响[J]．劳动科学，1979，55(12)：682-688
[43] 伊板等．高电界の生体系の影响[J]．静电气学会志，1975，(9)：176
[44] 唯薇，丁晓东，霍俊伟．草原樱桃与欧洲甜樱桃远缘杂交不亲和原因初探[J]．东北农业大学学报30(2)：148-153
[45] 万贤国．电离辐射技术在作物远缘杂交上的应用[J]．湖南农业科学，1994(3)：45-47
[46] 李玉晖，陈学森，郑洲．果树远缘杂交育种研究进展[J]．山东农业人学学报(自然科学版)，2003，34(1)：139-143
[47] 工树彦．石锦凤．李莉等．禾本科小麦族植物远缘杂交的障碍及其克服[J]．中国草地，2003，25(2)：59-62
[48] 宋道军，陈若雷，尹若春等．离子束介导植物分子超远缘杂交研究[J]．自然科学进展，2001，11(3)：327-330
[49] 苏学合，高国强，时香玉等．利用花粉辐照技术进行棉花种间远缘杂交研究[J]．山东农业科学，2000(2)：8-10
[50] 张国庆，周伟军，姚先伶等．芸薹属植物远缘杂交研究现状[J]．山西农业科学2001，29(4)：25-30
[51] 刘琳莉，强胜，宋小玲等．用荧光显微镜技术观察药用野生稻(Oryza officinalis Wall)和转基因水稻的不亲和性[J]．中国农业科学2004，37(4)：469-472
[52] 邢少辰，周开达，蔡玉红，植物远缘杂交的研究[J]．吉林农业科学，2000，25(4)：18-21
[53] 高新起，王秀玲．植物远缘杂交的障碍及其克服[J]．生物学通报，1998，33(12)
[54] 王幼平，罗鹏，何兴金．诸葛菜与芸苔属属间杂交时花粉-雌蕊相互作用的研究[J]．广 西植物，1997，17(4)：371-374
[55] 向旭．影响荔枝花粉活力的化学因子研究[J]．广东科学农业．2000．(06)：56-60
[56] 郭光明，张福锁，尚忠林．硼对百合花粉萌发过程中细胞内游离钙离子的影响[J]．中国农业大学学报2002，7(5)：32-37
[57] 王玉芹，刘昌智，邓洪民．应用10B研究不同油菜品种花器官的硼素基因型差异[J]．中国油料，1993，(4)：11-14
[58] 沈康，沈振国，徐汉卿等．油菜硼素营养与结实性的研究[J]．作物学报，1993，19(6)：539-545
[59] 褚天铎，陈家驹，刘昌智等．油菜缺硼花而不实原因的探讨[J]．植物营养与肥料学报，1996，2(1)：23-31
[60] 任沪生，陈仲西，肖昌珍．油菜萎缩不实病防治研究．中国科学院微量元素学术交流会汇刊[M]．北京：科学出版社，1980：87-98
[61] 张秀省，沈振国，沈康．硼对油菜花器官发育和结实性的影响[J]．土壤学报，1994，31(2)：146-152
[62] 沈振国，张秀省，王振宇等．硼素营养对油菜花粉萌发的影响[J]．中国农业学，1994，27(1)：51-56
[63] 杨弘远．钙在有花植物受精过程中的作用[J]．植物学报1999，41(10)：1027-1035
[64] 唐前瑞，陈友云．连蕊茶花粉发芽适宜培养基的探讨[J]．湖南林业科技．2004．31．(1)：32-35
[65] 薛妙男，刘华英．沙田柚花粉低温贮藏的研究[J]．广西植物．2000．20(4)：367-370
[66] 张玉进，张兴国．魔芋花粉的低温和超低温保存[J]．园艺学报．2000．27(2)：139-140
[67] 周莉，代力民，徐杰．丁香花粉生命力及贮藏力的研究[J]．沈阳农业大学学报，2003-02．34(1)：16-19
[68] F.奥斯伯，R.布伦特，R.E.金斯顿等．精编分子生物学指南[M]．北京：科学出版社，1998：120-130
[69] 崔大祥，闰小君，苏成芝．差异表达基因克隆技术的进展[J]．生命的化学，1999(5)
[70] Bauer D,Muller H,Reich J.et al.Identification of differentially expressed mRNA species by an improved display technique(DDRT-PCR)[J].Nucleic Acids Res,1993, 21 (18):4272-4280
[71] Ito T, Kito K, Adati N,etal.Fluorescent differential display: Arbitraryly primed RT-PCR fingerprinting on an automated .DNA sequencer[J].FEBE Letters,1994,35(23):231-236
[72] Liang P ,Zhu WM,Zhang XY, et al.Differential display using one-base anchored oligo-dT primers[J].Nucleic Acids Res,1994,22(25):5763-5764
[73] 陈斌，袁建刚，潘美辉等．差异显示反转录PCR技术的建立和改进[J]．基础医学与临床，1996：63-67
[74] Callard D, Lescure B, Mazzolini L. A method for the elimination of false positives generated by them. RNA differential display technique[J]. Bio Techniques, 1994(16): 1096-1103
[75] Corton JC, Gustafsson JA. Increased efficiency in screening large numbers of cDNA fragments generated by differential display[J]. Biotechniques, 1997. 22(5): 802-804. 806. 808
[76] Doss RP. Differential display without radioactivity-A modified procedure[J]. Bio Techniques 1996, 21(3): 408-412
[77] Gottschlich S, Goeroegh T, Folz BJ, et al. Optimized differential display and reamplification parameters for silverstaining[J]. Res Comm Mol Path Pharm, 1997, 97 (2): 237-240
[78] Hadman M, Adam BL, Wright GL, et al. Modification to the differential display technique reduces background and increase sensitivity[J]. Anal Biochem, 1995, 226: 383-386
[79] Ito T, Kito K, AdatiN, et al. Fluorescent differential display: Arbitraryly primed RT-PCR fingerprinting on an automated DNA sequencer[J]. FEBE Letters, 1994, 35(23): 231-236
[80] Ito T, Sakaki Y. Fluorescent differential display method for high speed scanning of tissue-or cell-specific transcripts[J]. Metods Mol Genet. 1996, 8: 229-245
[81] Liang P, Averboukh L, Pardee AB. Distribution and cloning of eukaryotic mRNAs by means of differential display: Refinements and optimization[J]. Nucleic Acids Res, 1993, 21: 3269-3275
[82] Liang P, Pardee AB. Differential display methods and protocols (methods in Molecular Biology, Vol. 85)[M]. Totowa, VewJersey: HumanaPress, I997, 45-103
[83] Liang Peng, Arthur B Pardee. Differential display of eukaryotic messenger RNA by means of the polymerase chain reaction[J]. Science, 1992, 257(14): 967-971
[84] Liu C, Raghothame KG. Practical method for cloning cDNAs generated in an mRNA differential display[J]. Bio Techniques, 1996, 20(4): 576-580
[85] Lohmann J, Schickle H, Bosch T C G. REN display, a rapid and efficient method for noradioactive differential display and mRNA isolation[J]. BioTchniques, 1995, 18(2): 200-202
[86] Luce MJ. Minimizing false positives in differential display[J]. Bio Techniques 1998. 24 (5): 766-768
[87] Malhotra K, Foltz L, Mahoney WC, et al. Interaction and effect of annealing temperature on primers used in differential, display RT-PCR[J]. Nucleic Acids Res, 1998, 26 (3): 854-856
[88] Matz MV, Lukyanov SA. Different strategies of differential display: areas of application [J].Nucleic Acids Res,1998,6(24):5537-5543
[89] Miele G.Elimination of false positives generated through PCR-reamplification of differential display cDNA[J].Bio techniques,1998,25(1):138-144
[90] Mol L, Miller HM,Li J,et al. Improvements to the differential display method for gene analysis[J]. Bio Biophysical Res Communications, 1994,199(2):564-569
[91] Schneiderbauer A, Sandermann H Jr, Ernst D.Isolation of functional RNA from plant tissues rich in phenolic compounds[J]. Anal Biochern,1991,(197):91-95
[92] Lewinsohu E. Steele CL, Croteau R. Simple isolation of functional RNA from woody stems of gymnosperms[J]. Plant Mol Biol Rep,1994,(12):20-25
[93] 王立秋．激光诱变的生物学效应及其在动植物遗传育种上的应用[J]．激光生物学报，1997．6(2)：1097-1102
[94] 刘家克．中国蔬菜栽培学[M]．中国农业科学院蔬菜研究所主编．农业出版社．1988：48-49
[95] 王玉成，薄海侠，杨传平．胡杨、柽柳总RNA提取方法的建立[J]．东北林业大学学报，2003，31(5)：99-100
[96] 徐祖荫，孙启跃，李干洲．不同破壁法对花粉提取液中可溶性营养成分的影响[J]．蜜蜂杂志月刊，1994，(1)：6-10
[97] 姚成义，赵洁．钙和硼对蓝猪耳花粉萌发及花粉管生长的影响[J]．武汉植物学研究，2004，22(1)：1-7
[98] 唐维，张星海．花粉破壁方法的研究进展[J]．食品与发酵工业，29(2)：86-92
[99] Kong Hai-Yan, Jia Gui-Xia.Calcium Distribution During Pollen Development of Larixprincipis-rupprechtii[J],植物学报，2004，46(7):60-76
[100] 李安平，谢碧霞，常银子．马尾松花粉破壁技术及其发酵饮料[J]．东北林业大学学报，32(1)，11-13