水稻Osg6B启动子的序列和功能分析及大肠杆菌argE基因的克隆和对烟草的转化
摘要
本项研究分为两个部分：“水稻启动子的序列和功能分析”和“大肠杆菌argE的克隆及对烟草的转化”。
第一部分：对已克隆的启动子Osg6B’进行的序列分析表明，Osg6B’全长1688bp，与报道的Osg6B有98％的同源性，两者在转录起始位点、TATA box及其它花药特异性启动子共有的保守序列TGTGG完全相同。但是与报道的Osg6B比较，在决定时空特异性的0～-1273bp功能区域内，有18处出现18碱基替换，18处出现22碱基缺失，3处出现3碱基插入；在核心功能序列区域(-1095bp～-1273bp)内，有3处出现3碱基替换，6处出现6碱基缺失。在序列分析的基础上，对Os96B’在转基因植株中的时空特异性进行了分析。将克隆的启动子与报告基因GUS相连，构建植物表达载体，通过农杆菌介导转化烟草。T_0代成熟植株中，在花药发育的不同时期和不同部位，以及在转基因植株的萼片、花瓣、柱头、花丝、花粉都检测出GUS活性。在9棵T_0幼苗植株中，2棵检测为阴性，4棵在根、茎、叶中检测出GUS活性，3棵在茎、叶中检测出GUS活性。3／4的T_1代总数的抗性幼苗的根、茎、叶检测到GUS活性。外源基因的遗传分析表明，T_1代的表型分离符合孟德尔法则。研究表明，所克隆的Osg6B’不具有花药特异性，而具有组成型表达的特征。
第二部分：从大肠杆菌E．coli中克隆出argE基因，全长1152bp,推导编码总数383个氨基酸的N-乙酰-鸟氨酸酶(N-acetylornithinase，NAO)。与报道的argE序列相比，DNA序列和推导的氨基酸序列完全相同。将argE与组成型启动子CaMV 35S相连，构建植物表达载体，通过农杆菌介导转化烟草。经过GUS染色、PCR及Southern鉴定获得了转基因植株。argE基因在转基因植株中的功能研究正在进行。
This study consisted of two parts: sequence and functional analysis of the cloned promoter Osg6B' from rice and molecular cloning and transferring into tobacco ofargE from E.coli.
In the part I, the sequence of the cloned promoter Osg6B' was first analyzed. Osg6B' had a whole length of 1688 bp and 98% homology to known sequence of promoter Osg6B. Its transcriptional start point, TATA boxes and the consensus sequences "TGTGG" conserved usually in anther-specific promoters were identical to those of reported Osg6B. However, the cloned promoter had 18 substitution at 18 sites, 22 deletions at 18 sites and 3 insertion at 3 sites between sites 0---1273 bp which was reported to control temporal-spatial specific expression , and 3 substitution at 3 sites , 6 deletions at 6 sites between -1095 bp and -1273 bp, the key functional sequence area, in comparing with known Osg6B. Based on the sequence analysis, the temporal-spatial speciality of Osg6B' was then analyzed with transgenic tobacco plants. A binary plant expression vector with Osg6B' driving report gene GUS was constructed and transferred into tobacco via Agrobacterium tumefaciens mediation. In TO mature transgenic plants tested, GUS st
aining was observed in different development stages and positions of the anther and some times in calyx, petal, stigma, anther filement and pollens. In 9 TO young plants GUS-tested, 2 plants were negative and the rest were positive: 4 plants stained in root, stem and leaves and 3 plants only in stem and leaves. GUS staining was also found in root, stem and leaves of Kan-resistant Tl seedlings that occurred 3/4 of all Tl generation. These results indicated that the cloned Osg6B' had a constitutive characteristics but not anther specificity, and the transgene was segregated in a Mendelian fashion in the Tl generation.
In the part II, a gene, argE, was cloned from E.coli and sequenced. It had 1152 bp in length, and predictably encoded for Af-acetylornithinase (NAO) with total 383 amino acids. Its DNA sequence and predicated amino acid sequence were entirely identical to those of reported argE. This gene, artificially put under the control of CaMV 35S, was introduced into tobacco with the aid of Agrobacterium, and the transgenic plants obtained were identified by GUS staining, PCR and Southern blot analysis. Its function in transgenic plants is being exploited.
第一部分：对已克隆的启动子Osg6B’进行的序列分析表明，Osg6B’全长1688bp，与报道的Osg6B有98％的同源性，两者在转录起始位点、TATA box及其它花药特异性启动子共有的保守序列TGTGG完全相同。但是与报道的Osg6B比较，在决定时空特异性的0～-1273bp功能区域内，有18处出现18碱基替换，18处出现22碱基缺失，3处出现3碱基插入；在核心功能序列区域(-1095bp～-1273bp)内，有3处出现3碱基替换，6处出现6碱基缺失。在序列分析的基础上，对Os96B’在转基因植株中的时空特异性进行了分析。将克隆的启动子与报告基因GUS相连，构建植物表达载体，通过农杆菌介导转化烟草。T_0代成熟植株中，在花药发育的不同时期和不同部位，以及在转基因植株的萼片、花瓣、柱头、花丝、花粉都检测出GUS活性。在9棵T_0幼苗植株中，2棵检测为阴性，4棵在根、茎、叶中检测出GUS活性，3棵在茎、叶中检测出GUS活性。3／4的T_1代总数的抗性幼苗的根、茎、叶检测到GUS活性。外源基因的遗传分析表明，T_1代的表型分离符合孟德尔法则。研究表明，所克隆的Osg6B’不具有花药特异性，而具有组成型表达的特征。
第二部分：从大肠杆菌E．coli中克隆出argE基因，全长1152bp,推导编码总数383个氨基酸的N-乙酰-鸟氨酸酶(N-acetylornithinase，NAO)。与报道的argE序列相比，DNA序列和推导的氨基酸序列完全相同。将argE与组成型启动子CaMV 35S相连，构建植物表达载体，通过农杆菌介导转化烟草。经过GUS染色、PCR及Southern鉴定获得了转基因植株。argE基因在转基因植株中的功能研究正在进行。
This study consisted of two parts: sequence and functional analysis of the cloned promoter Osg6B' from rice and molecular cloning and transferring into tobacco ofargE from E.coli.
In the part I, the sequence of the cloned promoter Osg6B' was first analyzed. Osg6B' had a whole length of 1688 bp and 98% homology to known sequence of promoter Osg6B. Its transcriptional start point, TATA boxes and the consensus sequences "TGTGG" conserved usually in anther-specific promoters were identical to those of reported Osg6B. However, the cloned promoter had 18 substitution at 18 sites, 22 deletions at 18 sites and 3 insertion at 3 sites between sites 0---1273 bp which was reported to control temporal-spatial specific expression , and 3 substitution at 3 sites , 6 deletions at 6 sites between -1095 bp and -1273 bp, the key functional sequence area, in comparing with known Osg6B. Based on the sequence analysis, the temporal-spatial speciality of Osg6B' was then analyzed with transgenic tobacco plants. A binary plant expression vector with Osg6B' driving report gene GUS was constructed and transferred into tobacco via Agrobacterium tumefaciens mediation. In TO mature transgenic plants tested, GUS st
aining was observed in different development stages and positions of the anther and some times in calyx, petal, stigma, anther filement and pollens. In 9 TO young plants GUS-tested, 2 plants were negative and the rest were positive: 4 plants stained in root, stem and leaves and 3 plants only in stem and leaves. GUS staining was also found in root, stem and leaves of Kan-resistant Tl seedlings that occurred 3/4 of all Tl generation. These results indicated that the cloned Osg6B' had a constitutive characteristics but not anther specificity, and the transgene was segregated in a Mendelian fashion in the Tl generation.
In the part II, a gene, argE, was cloned from E.coli and sequenced. It had 1152 bp in length, and predictably encoded for Af-acetylornithinase (NAO) with total 383 amino acids. Its DNA sequence and predicated amino acid sequence were entirely identical to those of reported argE. This gene, artificially put under the control of CaMV 35S, was introduced into tobacco with the aid of Agrobacterium, and the transgenic plants obtained were identified by GUS staining, PCR and Southern blot analysis. Its function in transgenic plants is being exploited.
引文
1．丁建清，外来种对中国生物多样性的影响，见《中国生物多样性国情研究报告》编写组(编)，中国生物多样性国情研究报告，北京：中国环境科学出版社，1998，58-61
2．葛盛军，金海平，紫茎泽兰的危害和防治，四川农业科技，2003(2)
3．郭光沁，郑国锠，绿色荧光蛋白在活体细胞分子生物学研究中的应用，世界科技研究与发展1998年，1：70-75
4．郭光远，杨宇容，马俊，刘勇，徐丽华，姜成林，国内新病害——紫茎泽兰叶斑病菌的研究，植物病理学报，1991，21(4)：245-250
5．何大愚，刘伦辉，紫茎泽兰防治研究进展，生态学进展，1988，5(3)：163-168
6．胡适宜．被子植物胚胎学．北京：高等教育出版社，1982，pp23～27
7．江家富，杂草生物防治研究回顾与展望(综叙)，安徽农业大学，2003,30(1)：61-65
8．金银根，生物工程技术在杂草治理中的应用，杂草科学，1999(3)：2-4
9．李胜国，刘玉乐，朱峰等．基因工程雄性不育烟草的获得．植物学报，1995，37(8)：659～660
10．李胜国，刘玉乐，田波．植物花粉发育的分子生物学研究进展．生物工程进展，1997，17：17～22
11．李孙荣，倪汉文，我国杂草防治进展与前景，作物杂志；1997(4)：5-6
12．刘长栋，抗除草剂作物对杂草科学的影响，农药科学与管理，2000，21(4)：32-34
13．陆桂华，张景六，洪孟民，RTS启动子与GUS的嵌合基因在转基因水稻中的专一性表达，植物生理学报，2000，26(2)：164-170
14．黄宝华，豚草在国内的分布及危害调查，植物检疫，1985，(1)：62-65
15．强胜，世界性恶性杂草—紫茎泽兰研究的历史及现状，武汉植物学研究，1998a，16(4)：354-360
16．强胜，曹学章，外来杂草在我国的危害性及其管理对策，生物多样性，2001，9(2)：188-195
17．桑卫国，我国外来杂草入侵特点的初步分析中国农业科技导报，2002，4(5)：59-61
18 佘宇平，梁家社，何大愚，紫茎泽兰防除方法的进展，杂草学报，1988，2(2)：39-40
19．苏少泉，杂草防治的发展趋向，世界农业，1996(7)：30-31
20．余炳生，张仪，生物学显微技术，北京农业大学(中国农业大学)出版社，1989，PP：1-47
21．余柳青，稻田畦畔和灌溉沟渠植物的多样性研究，农业植物学学术研讨会论文集，1997，4(1)：10-13
22．万方浩，王韧，豚草生物防治概况，杂草科学，1990，(22)：30-32
23．王峰，张晓俊，胡奇勇，陈莎莎，转基因作物与生物安全，福建农业学报，2000，15(增刊)：149-152
24．王关林，方宏筠，植物基因工程原理与技术，1998，知识出版社，pp：22-25
25．王宏富，抗除草剂转基因作物的潜在风险及其防范策略，中国农学通报，2001，vol17(4)：63-64
26．王灶安，植物学实验图说(解剖部分)，农业出版社，1992，pp：75-76
27．杨蓉西，危害严重的外来入侵植物—紫茎泽兰，生物学通报，2003，38(1)：20-21
28．张智英，魏艺，何大愚，泽兰实蝇生物学特性的初步研究，生物防治通报，1988，4(1)：10-13
29．赵国晶，云南省紫茎泽兰的分布与危害的调查研究，杂草学报，1989,3(2)：37-40；
30．郑宏红，瞿礼嘉，刘美华，张毅，顾红雅，陈章良，花药特异性表达的类查尔酮基因D5与水稻花粉发育有关，科学通报，2000，45(11)：1132-1137
31． Albani D, Robert L S, Donaldson P A et al. Characterization of a pollen-specific gene family from Brassica napus which is activated during early microspore development. Plant Mol. Biol., 1990, 15: 605-622
32． Albani D, Sardana R, Robert L S et al. A Brassica napus gene family which shows sequence similarityto ascorbate oxidase is expressed in developing pollen. Molecular characterization and analysis of a promoter activity in transgenic tobacco plants. Plant J., 1992, 2 (3) : 331-342
33． Altschul, Stephen F., Thomas L. Madden, Alejandro A. Schaffer, Jinghui Zhang, Zheng Zhang, Webb Miller, and David J. Lipman, Gapped BLAST and PSI-BLAST: a new generation of protein database search programs , Nucleic Acids Res. 1997, 25:3389-3402．
34． Atanassov I, Russinova E, Antonov L et al. Expression of an anther-specific chalcone synthase-like gene is correlated with uninucleate microspore development in Nicotiana sylvestis. Plant Mol. Biol., 1998, 38 (6) : 1169-1178
35． Bate N, Twell D. Functional architecture of a late pollen promoter: pollen-specific transcription is developmentally regulated by multiple stage-specific and co-dependent activator elements. Plant Mol. Biol., 1998, 37 (5) : 859-869
36． Beerhues L, Forkmann G, Schopker H et al. Flavanones 3-hydroxylase and dihydroflavonol oxygenase activities in anthers of tulipa. The significance of the tapetum formation in flavonoid metabolism. J. Plant Physiol., 1989, 133: 743-746
37． Bengtsson B. Prons and cons of foreign genes in crops. Nature, 1997, 385: 290
38． Carpenter J L, Ploense S E, Snustad D P et al. Preferential expression of an alpha-tubalin gene of Arabidopsis in pollen. Plant Cell, 1992,4 (5) : 557-571
39． Chalfie, M., Tu, Y., Euskirchen, G, Ward, W. and Prasher, D.,. Green fluorescent protein as a marker for gene expression. Science. 1994,263: 802-5．
40． Crabeel,M.,Charlier,D.,Studies on the bipolar argECBH operon of E.coli characterization of restriction endonuclease fragments obtained from A. dargEBCH transducing phages and a colE1 argEBCH plasmid MOL.Gen.Genet. 1977(151) :161-168
41． Cline J, Braman J C, Hogrefe H H. PCR fidelity of Pfu DNA polymerase and other thermastable DNA polymerase. Nucleic Acids Res., 1996, 24 (18) : 3546-3551
42． Davis,R.H..Compartmental and regulatory mechanisms in the arginine pathways of Neurospora crassa and Saccharomyces cerevisiae. Microbiol.Rev. 1986,50:280-313
43． Droge W, Broer I, Transgenic plants containing the phosphinothricin-N-acetyltransferase gene metabolize the herbicide L-phosphinothricin (glufosinate) differently from untransformed plants. PLANTA ,1992(187) :142-151
44． Droge W, Broer I, The metabolites of the herbicide L-phosphinothricin (glufosinate) identification , stability and mobility in transgenic , herbicide resistant an untransformed plants. Plant Physiology, 1994(105) :pp 159-166
45． Dzelzkalns V A, Thorsness M K, Dwyer K G et al. Distinct cis-acting elements direct pistil-specific and pollen-specific activity of the Brassica S Locus Glycoprotein gene promoter. Plant Cell, 1993, 5
(8) : 855-863
46． Flaman J M, Frebourg T, Moreau V et al. Nucleic Acids Res., 1994, 22: 3259-3260
47． Fuerstenberg S I, Bucciaglia P A, Smith A G Molecular characterization of an anther-sfecific gene from tobacco shows seqence similarity to a tapetum-specific gene from tomato. Sex. Plant Reprod., 2000, 12 (4) : 250-252
48． Gell AC, Bacic A,1986,Arabinogalactan-proteins of the female sexual tissue of Nicotiana alata. Plant physiol 82:885-889
49． Goldberg R B, Beals T P, Sanders P M. Anther development: Basic principles and practical applications. Plant Cell, 1993,5: 1217-1219
50． Guerrero F D, Crossland L, Smutzer G S et al. Promoter seqences from a maize pollen-specific gene direct tissue-specific transcription in tobacco. Mol. Gen. Genet., 1990, 224: 161-168
51． Hamilton D A, Roy M, Rueda J et al. Dissection of a pollen-specific promoter from maize by transient transformation assays. Plant Mol. Biol., 1992, 18:211-218
52． Hamilton D A, Schwarz Y H, Mascarenhas J P. A monocot pollen-specific promoter contains separable pollen-specific and quantitative elements. Plant Mol. Biol., 1998, 38 (4) : 663-669
53． Hamilton D A, Schwarz Y H, Rueda J et al. Comparison of transient stable expression by a pollen-specific promoter: the transformation results do not always agree. Sex. Plant Reprod., 1999,12 (5) : 292-295
54． Jefferson R A, Kavangh T A, Bevan M W. GUS fusion: β-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J., 1987,6:3901-3907
55． Jeon J S, Chuang Y Y, Lee S C et al. Isolation and characterization of an anther-spscific gene, RA8, from rice (Oryza sativa L.). Plant Mol. Biol. 1999,39 (1) : 35-44
56． Meinnel.T, Structural and Biochemical Characterization of the Escherichia coli argE Gene Product. J. Bacteriology, Apr. 1992,p.2323-2331
57． Kamalay J C, Goldberg R B. Regulation of structural gene expression in tobacco. Cell, 1980,19: 935-446
58． Kamalay J C, Goldberg R B. Organ-specific nuclear RNAs in tobacco. Proc. Natl. Acad. Sci. USA, 1984,81:2801-2805
59． Kim H U, Chung T U. Characterization of three anther-specific genes isolated from Chinese cabbage.Plant Mol. Biol., 1997,33 (1) : 193-198
60． Knox RB,1984, Pollen-pistil interactions .In :Linskens HF. Heslop-Harrison J (eds) Cellular interactions .Encycl Plant Physiol 17:508-608
61． Koltunow A M, Truettner J, Cox K H et al. Different temporal and spatial expression patterns occur during anther development. Plant Cell, 1990, 2: 1201-1224
62． Kriete .G ,Niehaus .K ,A .M. Perlick, Puhler. A ,Broer .I, Male sterility in transgenic tobacco plants induced by tapetum-specific deacetylation of the externally applied non-toxic compound N-acetyl-L-phosphinothricin ,The Plant Journa,1996,9(6) :809-818
63． Leon Plegt and Raoul J .Bino, β-Glucuronddase activity during development of the male
gametophyte from transgenic and non-transgenic plants.,Mol Gen Genet,1989,216:321-327
64． Lee JY, Aledmita RR,Hodges TK(1996) .Isolation of a tapetum-specific gene and promoter from rice. Int Rice Res Notes,21 (2/3) :37-38
65． Mascareenhas J P. Gene activity during pollen development. Annu. Rev. Plant Physiol., 1990, 41: 317-338
66． Mascareenhas J P. Pollen expressed genes and their regulations. In: Angiosperm pollen and ovules. Springer-Verlag. 1992, 1-5
67． McCormick S. Molecular analysis of male gametogenesis in plant. Trends Genet., 1991, 7: 298-303
68． Meinnel.T(1992) , Structural and Biochemical Characterization of the Escherichia coli argE Gene Product. J. Bacteriology, Apr. 1992,p.2323-2331
69． Mullis K B, Ferre F, Gibbs R A. The polymerase chain reaction (陈受宜，朱立煌等译), Birkhauser Boston, 1994, 4
70． Paul W, Hodge R, Smartt S et al. The isolation and characterisation of the tapetum-specific Arobidopsis thaliana A9) gene. Plant Mol. Biol., 1992, 19: 611-622
71． Qiang S and Summerell B A,Pathogenicity of Alternaria alternate on crofton weed (Eupatorium adenophorum).In:Proceedings of 17th Asian-Pacific Weed Science Society Conference.Bangkok:The Organization of the 17th Asian-Pacific Weed Science Society Conference, 1999,345-347
72． Rieger M. A .,et al. Risks of gene flow from transgenic herbicide-sesistant canola (Brassica napus) to weedy relatives in southern Australian cropping systems , Aust J Agric. Res., 1999,50:115-128
73． Sakamoto M, Sanada Y,Tagiri A, Murakami T, Ohashi Y, Matsuoka M, Structure and characterization of a gene for light-harvesting Chl a/b binding protein from rice .Plant Cell Physiol ,1991,32:385-393
74． Sambrook. J, David Russell, Molecular Cloning: A Laboratry Manual (Ⅲ), Cold Spring Harbor Lab(CSHL) Press, 2001 ;黄培堂等译，科学出版社，2002年9月出版
75． Sari Gorla G, Frovac C, Binelli A et al. The extent of gametophytic-sporophytic gene expression in maize. Theor. Appl. Genet., 1986, 72: 42-47
76． Schmulling T et al. Promoters of the rol A,B and C genes of Agrobacterium rhizogenes are differentially regulated in transgenic plants .Plant Cell, 1989,1(7) :665-670
77． Scortt R, Hodge R, Paul W et al. The molecullar biology anther differentiation. Plant Sci., 1991, 80: 167-191
78． Spena A et al. The plant oncogenes rol A,B and C from Agrobacterium rhizogenes :Effects on morphology ,development and hormone metabolism. Method Mol Biol, 1995,44:207-222
79． Staiger D, Kappeler S, Muller M et al. The proteins encoded by two tapetum-specific transcripts, Satap35 and Satap44, from Sinapis alba L. are located in the exine cell wall layers of developing microspores. Planta, 1994, 192: 221-231
80． Takanori Sato, Mary K.Thorsness, Muthugapatti K.Kandasamy , Takeshi Nishio ,Masashi Hiral ,June B.Nasrallah, and Mikhail E.Nasrallah ,Activity of an S Locus gene promoter in pistils and anthers of transgenic Brassica ,The Plant Cell, 1991,3:867-876
81． Tanksley S D, Zamir D, Rick C M. Evidence for extensive overlap of sporophytic and gametophytic gene expression in Lycopersicon esculentum. Science, 1981, 213: 453-455
82． Thorsness, M.K., Kandasamy ,M.K.,Nasrallah ,M.E.,and Nasrallah , J.B., A Brassica S-locus gene promoter targets toxic gene expression and cell death to the pistil and pollen of transgenic Nicotiana. Dev.Biol.1991,143:173-184
83． Toriyama, K.,Thorsness ,M.K., Nasrallah,J.B.N and Nasrallah, M.E. A Brassica S-locus gene promoter directs sporophytic expression in the anther tapetum of transgenic Arabidopsis, Dev.Biol. 1991,143:427-431
84． Treacy B K, Hattori-J, Prud-homme, I et al. Bnm1, a Brassica pollen-specific gene. Plant Mol. Biol., 1997, 34 (4) : 603-611
85． Tsuchiya T, Toriyama K, Nasrallah M E et al. Isolation of genes abundently expressed in rice anthers at the microspore stage. Plant Mol. Biol., 1992, 20: 1189-1193
86． Tsuchiya T, Toriyama K, Ejiri S et al. Molecular characterization of rice genes specifically expressed in the anther tapetum. Plant Mol. Biol. 1994,26:1737-1746
87． Tsuchiya T, Toriyama K, Hinata K et al. Tapetum-specific expression of the Osg6B promoter-β-glucuronidase gene in transgenic rice. Plant Cell Rep., 1997,16: 363-367
88． Twell D, Yamaguchi J, McCormick S. Pollen-specific gene expression in transgenic plants: Coordinate regulation of two different tomato gene promoters during microsporogenesis. Genes Dev., 1990, 109: 705-713
89． Twell D, Yamaguchi J, Wing R A et al. Promoter analysis of genes that are coordinately expressed during pollen development reveals pollen-specific enhancer sequences and shared regulatory elements.Genes Dev., 1991,5:496-507
90． Van der Meer I M, Stam M E, van Tunen A J et al. Antisense inhibition of flavonoid biosynthesis in Petunia anthers results in male sterility. Plant Cell, 1992,4:253-262
91． Van Tunen A J, Hartman S A, Mur L A et al. Regulation of chalcone isomerase (CHI) gene expression in Petunia hybrida: the use of alternative promoters in corolla, anthers and pollen. Plant Mol. Biol., 1989,12: 539-551
92． Willing R P, Mascarenhas J P. Analysis of the complexity and diversity of mRNAs from pollen and shoots of Tradescantia. Plant Physiol., 1984, 75: 865-868
93． Willing R P, Bashe D, Mascarenhas J P et al. An analysis of the quantity and diversity of messenger RNAs from pollen and shoots of Zea mays. Theor. Appl. Genet., 1988, 75: 751-753
94． Wittink F R A, Knuiman B, Derkson J et al. The pollen-specific gene Ntp303 encodes a 69-kDa glycoprotein associated with the vegetative memberanes and the cell wall. Sex Plant Reprod., 1999, 12(5) : 276-284
95． Xu H ,Davies SP ,Kwan BYH ,O'Brien AP, Singh M ,Knox RB , Haploid and diploid expression of a Brassica campestris anther-specific gene promoter in Arabidopsis and tobacco, Mol Gen Genet, 1993,239:58-65
96． Zabaleta E, Heiser V, Grohmann L et al. Promoters of nuclear-encoded respiratory chain Complex I
genes from Arabidopsis thaliana contain a region essential for anther/pollen-specific expression. Plant J., 1998, 15(1) : 49-59
97． Zuo J R, Zhan X Y, Wu H M et al. Characterization of a rice pollen-specific gene and its expression. American J. Bot., 1994, 81(5) : 552-561
2．葛盛军，金海平，紫茎泽兰的危害和防治，四川农业科技，2003(2)
3．郭光沁，郑国锠，绿色荧光蛋白在活体细胞分子生物学研究中的应用，世界科技研究与发展1998年，1：70-75
4．郭光远，杨宇容，马俊，刘勇，徐丽华，姜成林，国内新病害——紫茎泽兰叶斑病菌的研究，植物病理学报，1991，21(4)：245-250
5．何大愚，刘伦辉，紫茎泽兰防治研究进展，生态学进展，1988，5(3)：163-168
6．胡适宜．被子植物胚胎学．北京：高等教育出版社，1982，pp23～27
7．江家富，杂草生物防治研究回顾与展望(综叙)，安徽农业大学，2003,30(1)：61-65
8．金银根，生物工程技术在杂草治理中的应用，杂草科学，1999(3)：2-4
9．李胜国，刘玉乐，朱峰等．基因工程雄性不育烟草的获得．植物学报，1995，37(8)：659～660
10．李胜国，刘玉乐，田波．植物花粉发育的分子生物学研究进展．生物工程进展，1997，17：17～22
11．李孙荣，倪汉文，我国杂草防治进展与前景，作物杂志；1997(4)：5-6
12．刘长栋，抗除草剂作物对杂草科学的影响，农药科学与管理，2000，21(4)：32-34
13．陆桂华，张景六，洪孟民，RTS启动子与GUS的嵌合基因在转基因水稻中的专一性表达，植物生理学报，2000，26(2)：164-170
14．黄宝华，豚草在国内的分布及危害调查，植物检疫，1985，(1)：62-65
15．强胜，世界性恶性杂草—紫茎泽兰研究的历史及现状，武汉植物学研究，1998a，16(4)：354-360
16．强胜，曹学章，外来杂草在我国的危害性及其管理对策，生物多样性，2001，9(2)：188-195
17．桑卫国，我国外来杂草入侵特点的初步分析中国农业科技导报，2002，4(5)：59-61
18 佘宇平，梁家社，何大愚，紫茎泽兰防除方法的进展，杂草学报，1988，2(2)：39-40
19．苏少泉，杂草防治的发展趋向，世界农业，1996(7)：30-31
20．余炳生，张仪，生物学显微技术，北京农业大学(中国农业大学)出版社，1989，PP：1-47
21．余柳青，稻田畦畔和灌溉沟渠植物的多样性研究，农业植物学学术研讨会论文集，1997，4(1)：10-13
22．万方浩，王韧，豚草生物防治概况，杂草科学，1990，(22)：30-32
23．王峰，张晓俊，胡奇勇，陈莎莎，转基因作物与生物安全，福建农业学报，2000，15(增刊)：149-152
24．王关林，方宏筠，植物基因工程原理与技术，1998，知识出版社，pp：22-25
25．王宏富，抗除草剂转基因作物的潜在风险及其防范策略，中国农学通报，2001，vol17(4)：63-64
26．王灶安，植物学实验图说(解剖部分)，农业出版社，1992，pp：75-76
27．杨蓉西，危害严重的外来入侵植物—紫茎泽兰，生物学通报，2003，38(1)：20-21
28．张智英，魏艺，何大愚，泽兰实蝇生物学特性的初步研究，生物防治通报，1988，4(1)：10-13
29．赵国晶，云南省紫茎泽兰的分布与危害的调查研究，杂草学报，1989,3(2)：37-40；
30．郑宏红，瞿礼嘉，刘美华，张毅，顾红雅，陈章良，花药特异性表达的类查尔酮基因D5与水稻花粉发育有关，科学通报，2000，45(11)：1132-1137
31． Albani D, Robert L S, Donaldson P A et al. Characterization of a pollen-specific gene family from Brassica napus which is activated during early microspore development. Plant Mol. Biol., 1990, 15: 605-622
32． Albani D, Sardana R, Robert L S et al. A Brassica napus gene family which shows sequence similarityto ascorbate oxidase is expressed in developing pollen. Molecular characterization and analysis of a promoter activity in transgenic tobacco plants. Plant J., 1992, 2 (3) : 331-342
33． Altschul, Stephen F., Thomas L. Madden, Alejandro A. Schaffer, Jinghui Zhang, Zheng Zhang, Webb Miller, and David J. Lipman, Gapped BLAST and PSI-BLAST: a new generation of protein database search programs , Nucleic Acids Res. 1997, 25:3389-3402．
34． Atanassov I, Russinova E, Antonov L et al. Expression of an anther-specific chalcone synthase-like gene is correlated with uninucleate microspore development in Nicotiana sylvestis. Plant Mol. Biol., 1998, 38 (6) : 1169-1178
35． Bate N, Twell D. Functional architecture of a late pollen promoter: pollen-specific transcription is developmentally regulated by multiple stage-specific and co-dependent activator elements. Plant Mol. Biol., 1998, 37 (5) : 859-869
36． Beerhues L, Forkmann G, Schopker H et al. Flavanones 3-hydroxylase and dihydroflavonol oxygenase activities in anthers of tulipa. The significance of the tapetum formation in flavonoid metabolism. J. Plant Physiol., 1989, 133: 743-746
37． Bengtsson B. Prons and cons of foreign genes in crops. Nature, 1997, 385: 290
38． Carpenter J L, Ploense S E, Snustad D P et al. Preferential expression of an alpha-tubalin gene of Arabidopsis in pollen. Plant Cell, 1992,4 (5) : 557-571
39． Chalfie, M., Tu, Y., Euskirchen, G, Ward, W. and Prasher, D.,. Green fluorescent protein as a marker for gene expression. Science. 1994,263: 802-5．
40． Crabeel,M.,Charlier,D.,Studies on the bipolar argECBH operon of E.coli characterization of restriction endonuclease fragments obtained from A. dargEBCH transducing phages and a colE1 argEBCH plasmid MOL.Gen.Genet. 1977(151) :161-168
41． Cline J, Braman J C, Hogrefe H H. PCR fidelity of Pfu DNA polymerase and other thermastable DNA polymerase. Nucleic Acids Res., 1996, 24 (18) : 3546-3551
42． Davis,R.H..Compartmental and regulatory mechanisms in the arginine pathways of Neurospora crassa and Saccharomyces cerevisiae. Microbiol.Rev. 1986,50:280-313
43． Droge W, Broer I, Transgenic plants containing the phosphinothricin-N-acetyltransferase gene metabolize the herbicide L-phosphinothricin (glufosinate) differently from untransformed plants. PLANTA ,1992(187) :142-151
44． Droge W, Broer I, The metabolites of the herbicide L-phosphinothricin (glufosinate) identification , stability and mobility in transgenic , herbicide resistant an untransformed plants. Plant Physiology, 1994(105) :pp 159-166
45． Dzelzkalns V A, Thorsness M K, Dwyer K G et al. Distinct cis-acting elements direct pistil-specific and pollen-specific activity of the Brassica S Locus Glycoprotein gene promoter. Plant Cell, 1993, 5
(8) : 855-863
46． Flaman J M, Frebourg T, Moreau V et al. Nucleic Acids Res., 1994, 22: 3259-3260
47． Fuerstenberg S I, Bucciaglia P A, Smith A G Molecular characterization of an anther-sfecific gene from tobacco shows seqence similarity to a tapetum-specific gene from tomato. Sex. Plant Reprod., 2000, 12 (4) : 250-252
48． Gell AC, Bacic A,1986,Arabinogalactan-proteins of the female sexual tissue of Nicotiana alata. Plant physiol 82:885-889
49． Goldberg R B, Beals T P, Sanders P M. Anther development: Basic principles and practical applications. Plant Cell, 1993,5: 1217-1219
50． Guerrero F D, Crossland L, Smutzer G S et al. Promoter seqences from a maize pollen-specific gene direct tissue-specific transcription in tobacco. Mol. Gen. Genet., 1990, 224: 161-168
51． Hamilton D A, Roy M, Rueda J et al. Dissection of a pollen-specific promoter from maize by transient transformation assays. Plant Mol. Biol., 1992, 18:211-218
52． Hamilton D A, Schwarz Y H, Mascarenhas J P. A monocot pollen-specific promoter contains separable pollen-specific and quantitative elements. Plant Mol. Biol., 1998, 38 (4) : 663-669
53． Hamilton D A, Schwarz Y H, Rueda J et al. Comparison of transient stable expression by a pollen-specific promoter: the transformation results do not always agree. Sex. Plant Reprod., 1999,12 (5) : 292-295
54． Jefferson R A, Kavangh T A, Bevan M W. GUS fusion: β-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J., 1987,6:3901-3907
55． Jeon J S, Chuang Y Y, Lee S C et al. Isolation and characterization of an anther-spscific gene, RA8, from rice (Oryza sativa L.). Plant Mol. Biol. 1999,39 (1) : 35-44
56． Meinnel.T, Structural and Biochemical Characterization of the Escherichia coli argE Gene Product. J. Bacteriology, Apr. 1992,p.2323-2331
57． Kamalay J C, Goldberg R B. Regulation of structural gene expression in tobacco. Cell, 1980,19: 935-446
58． Kamalay J C, Goldberg R B. Organ-specific nuclear RNAs in tobacco. Proc. Natl. Acad. Sci. USA, 1984,81:2801-2805
59． Kim H U, Chung T U. Characterization of three anther-specific genes isolated from Chinese cabbage.Plant Mol. Biol., 1997,33 (1) : 193-198
60． Knox RB,1984, Pollen-pistil interactions .In :Linskens HF. Heslop-Harrison J (eds) Cellular interactions .Encycl Plant Physiol 17:508-608
61． Koltunow A M, Truettner J, Cox K H et al. Different temporal and spatial expression patterns occur during anther development. Plant Cell, 1990, 2: 1201-1224
62． Kriete .G ,Niehaus .K ,A .M. Perlick, Puhler. A ,Broer .I, Male sterility in transgenic tobacco plants induced by tapetum-specific deacetylation of the externally applied non-toxic compound N-acetyl-L-phosphinothricin ,The Plant Journa,1996,9(6) :809-818
63． Leon Plegt and Raoul J .Bino, β-Glucuronddase activity during development of the male
gametophyte from transgenic and non-transgenic plants.,Mol Gen Genet,1989,216:321-327
64． Lee JY, Aledmita RR,Hodges TK(1996) .Isolation of a tapetum-specific gene and promoter from rice. Int Rice Res Notes,21 (2/3) :37-38
65． Mascareenhas J P. Gene activity during pollen development. Annu. Rev. Plant Physiol., 1990, 41: 317-338
66． Mascareenhas J P. Pollen expressed genes and their regulations. In: Angiosperm pollen and ovules. Springer-Verlag. 1992, 1-5
67． McCormick S. Molecular analysis of male gametogenesis in plant. Trends Genet., 1991, 7: 298-303
68． Meinnel.T(1992) , Structural and Biochemical Characterization of the Escherichia coli argE Gene Product. J. Bacteriology, Apr. 1992,p.2323-2331
69． Mullis K B, Ferre F, Gibbs R A. The polymerase chain reaction (陈受宜，朱立煌等译), Birkhauser Boston, 1994, 4
70． Paul W, Hodge R, Smartt S et al. The isolation and characterisation of the tapetum-specific Arobidopsis thaliana A9) gene. Plant Mol. Biol., 1992, 19: 611-622
71． Qiang S and Summerell B A,Pathogenicity of Alternaria alternate on crofton weed (Eupatorium adenophorum).In:Proceedings of 17th Asian-Pacific Weed Science Society Conference.Bangkok:The Organization of the 17th Asian-Pacific Weed Science Society Conference, 1999,345-347
72． Rieger M. A .,et al. Risks of gene flow from transgenic herbicide-sesistant canola (Brassica napus) to weedy relatives in southern Australian cropping systems , Aust J Agric. Res., 1999,50:115-128
73． Sakamoto M, Sanada Y,Tagiri A, Murakami T, Ohashi Y, Matsuoka M, Structure and characterization of a gene for light-harvesting Chl a/b binding protein from rice .Plant Cell Physiol ,1991,32:385-393
74． Sambrook. J, David Russell, Molecular Cloning: A Laboratry Manual (Ⅲ), Cold Spring Harbor Lab(CSHL) Press, 2001 ;黄培堂等译，科学出版社，2002年9月出版
75． Sari Gorla G, Frovac C, Binelli A et al. The extent of gametophytic-sporophytic gene expression in maize. Theor. Appl. Genet., 1986, 72: 42-47
76． Schmulling T et al. Promoters of the rol A,B and C genes of Agrobacterium rhizogenes are differentially regulated in transgenic plants .Plant Cell, 1989,1(7) :665-670
77． Scortt R, Hodge R, Paul W et al. The molecullar biology anther differentiation. Plant Sci., 1991, 80: 167-191
78． Spena A et al. The plant oncogenes rol A,B and C from Agrobacterium rhizogenes :Effects on morphology ,development and hormone metabolism. Method Mol Biol, 1995,44:207-222
79． Staiger D, Kappeler S, Muller M et al. The proteins encoded by two tapetum-specific transcripts, Satap35 and Satap44, from Sinapis alba L. are located in the exine cell wall layers of developing microspores. Planta, 1994, 192: 221-231
80． Takanori Sato, Mary K.Thorsness, Muthugapatti K.Kandasamy , Takeshi Nishio ,Masashi Hiral ,June B.Nasrallah, and Mikhail E.Nasrallah ,Activity of an S Locus gene promoter in pistils and anthers of transgenic Brassica ,The Plant Cell, 1991,3:867-876
81． Tanksley S D, Zamir D, Rick C M. Evidence for extensive overlap of sporophytic and gametophytic gene expression in Lycopersicon esculentum. Science, 1981, 213: 453-455
82． Thorsness, M.K., Kandasamy ,M.K.,Nasrallah ,M.E.,and Nasrallah , J.B., A Brassica S-locus gene promoter targets toxic gene expression and cell death to the pistil and pollen of transgenic Nicotiana. Dev.Biol.1991,143:173-184
83． Toriyama, K.,Thorsness ,M.K., Nasrallah,J.B.N and Nasrallah, M.E. A Brassica S-locus gene promoter directs sporophytic expression in the anther tapetum of transgenic Arabidopsis, Dev.Biol. 1991,143:427-431
84． Treacy B K, Hattori-J, Prud-homme, I et al. Bnm1, a Brassica pollen-specific gene. Plant Mol. Biol., 1997, 34 (4) : 603-611
85． Tsuchiya T, Toriyama K, Nasrallah M E et al. Isolation of genes abundently expressed in rice anthers at the microspore stage. Plant Mol. Biol., 1992, 20: 1189-1193
86． Tsuchiya T, Toriyama K, Ejiri S et al. Molecular characterization of rice genes specifically expressed in the anther tapetum. Plant Mol. Biol. 1994,26:1737-1746
87． Tsuchiya T, Toriyama K, Hinata K et al. Tapetum-specific expression of the Osg6B promoter-β-glucuronidase gene in transgenic rice. Plant Cell Rep., 1997,16: 363-367
88． Twell D, Yamaguchi J, McCormick S. Pollen-specific gene expression in transgenic plants: Coordinate regulation of two different tomato gene promoters during microsporogenesis. Genes Dev., 1990, 109: 705-713
89． Twell D, Yamaguchi J, Wing R A et al. Promoter analysis of genes that are coordinately expressed during pollen development reveals pollen-specific enhancer sequences and shared regulatory elements.Genes Dev., 1991,5:496-507
90． Van der Meer I M, Stam M E, van Tunen A J et al. Antisense inhibition of flavonoid biosynthesis in Petunia anthers results in male sterility. Plant Cell, 1992,4:253-262
91． Van Tunen A J, Hartman S A, Mur L A et al. Regulation of chalcone isomerase (CHI) gene expression in Petunia hybrida: the use of alternative promoters in corolla, anthers and pollen. Plant Mol. Biol., 1989,12: 539-551
92． Willing R P, Mascarenhas J P. Analysis of the complexity and diversity of mRNAs from pollen and shoots of Tradescantia. Plant Physiol., 1984, 75: 865-868
93． Willing R P, Bashe D, Mascarenhas J P et al. An analysis of the quantity and diversity of messenger RNAs from pollen and shoots of Zea mays. Theor. Appl. Genet., 1988, 75: 751-753
94． Wittink F R A, Knuiman B, Derkson J et al. The pollen-specific gene Ntp303 encodes a 69-kDa glycoprotein associated with the vegetative memberanes and the cell wall. Sex Plant Reprod., 1999, 12(5) : 276-284
95． Xu H ,Davies SP ,Kwan BYH ,O'Brien AP, Singh M ,Knox RB , Haploid and diploid expression of a Brassica campestris anther-specific gene promoter in Arabidopsis and tobacco, Mol Gen Genet, 1993,239:58-65
96． Zabaleta E, Heiser V, Grohmann L et al. Promoters of nuclear-encoded respiratory chain Complex I
genes from Arabidopsis thaliana contain a region essential for anther/pollen-specific expression. Plant J., 1998, 15(1) : 49-59
97． Zuo J R, Zhan X Y, Wu H M et al. Characterization of a rice pollen-specific gene and its expression. American J. Bot., 1994, 81(5) : 552-561