禽流感病毒(H5N1)基因(h5n1a)在马铃薯中的转化与表达研究——附：乙肝表面抗原(HBsAg)基因转化的对比研究
|
摘要
本研究首次将禽流感病毒（H_5N_1）基因h5n1a导入马铃薯获得
表达，并优化了转化系统，为商品化生产转基因植物疫苗提供科
学依据。
1、为了优化马铃薯（Solanum tuberosum L.）以块茎为受体
的再生系统。着重对诱导分化和结薯培养基作了较系统的筛选。
以MS为基本培养基，调整激素配比，得到M1、M2、M3、M4等4
种培养基。比较试验结果以M4（MS＋2.0mg／L BA＋3.0mg／L ZT＋0.5mg／L
NAA＋0.5mg／L GA_3）诱导块茎分化的效果最佳，丛芽分化频率达89.4％，
分化时间提早约20天。同时筛选了诱导结薯的较优培养方法，获
得使试管薯结薯数增加近1倍的壮苗和诱导结薯培养基组合（壮
苗：1／2MS＋0.5mg／L BA＋0.5mg／L GA_3＋3％Suc；诱导结薯：MS＋2.0mg／L
BA＋3.0mg／L ZT＋10％Suc），为获得高频转化奠定了基础。
2、构建了4个植物表达双元载体。p1301BG含有增强型绿色
莹光蛋白基因egfp；p1301BΩG在egfp与35s启动子之间插入65bp
的增强子序列Ω因子；p1301H5N1含有禽流感病毒基因h5n1a；
p1301HBs含有乙肝表面抗原基因HBsAg。这4个载体均以35s为
真核表达启动子，报告基因和目的基因以Tnos为终止子、植物筛
选标记基因以35s PolyA加尾，融合成嵌合基因。原核抗性为Kan。
分别用直接法导入农杆菌EHA105、LBA4404和AGL1。
3、初步比较了ZHFQ-2型火药式基因枪与农杆菌的转化效果，
发现，该基因枪微弹分散性差，转化结果不稳定，对受体材料损
伤较大，未能筛选到转化株；而农杆菌转化相对较稳定，并初步
发现外源基因的瞬时表达大多集中在受伤和形成层部位。
4、对农杆菌转化条件的比较研究发现，菌株、菌体状态、稀
中文摘要
释度以及真空、激素和AS处理，均对转化有很大影响。对3个菌
株（LBA4404、EHA105和AGLI）的侵染性比较发现，用EHA105转化
马铃薯块茎，GUS瞬时表达效果更好；比较几种转化条件下的GUS
瞬时表达结果，筛选到可获得高且稳定的GUS瞬时表达的转化条
件：菌体用 Mg（MS＋0．sing／L GA3＋10umol／L AS）液体培养基稀释
4倍，与受体于28oC、100rpm振荡侵染20－30min，共培养时在分
化培养基上加 30卜 mol／L AS（乙酚丁香酮），16hr弱光照共培养 3d，
使GUS瞬时表达活性提高了3．4倍。
5、在进行抗生素基础抗性测定和转化体筛选时，比较马铃薯块茎
对Hgg和Kan两种筛选剂的敏感性，发现马铃薯块茎受体对Hnn更
敏感，因此选用Hgg为抗性筛选剂。在此基础上改进了筛选策略，
提出间段压力筛选法。该方法与常规的连续压力筛选法相比，大
大减低了基础抗性确定的主观性对转化效率的影响。使获得抗性
株的频率提高了82儿外源基因阳性率提高了2．4倍，获得转化株
的时间提早7－10天。
6、获得 HSNI转基因马铃薯株系 31个，并对其外源基因的整
合和表达做了系统检测。GUS表达活性结果表明，外源基因己插入
马铃薯基因组并得到表达，但株系间差异较大，说明外源基因整
合的随机性。用hsnla双引物扩增马铃薯基因组总DNA，得到与
目的基因大小一致的特异性片段。以DIG标记的hsnla探针进行
Southern杂交，进一步证实基因hsnla已整合到马铃薯基因组中，
且初步确定为单拷贝插入。
7、用HSNI 鼠单抗与马铃薯块茎蛋白粗提液进行抗原抗体反
应证实，在马铃薯中表达的外源基因具有抗原抗体反应活性，这
是禽流感病毒（HSNI）基因首次在植物中的表达。由于禽流感病
2
禽流感病毒（HSNI）基因（hjnla）在马铃薯中的转化与表达研究
毒m）是新病毒株，最近才得到其鼠的单克隆抗体，还末能
进行定量检测，以及动物免疫实验结果检测。
8、为了证实所建立的转化系统的有效性，本研究同时进行了
乙肝表面抗原＊BSAg）基因在马铃薯中的表达研究，并对结果做
了详细报道。在马铃薯中表达的乙肝表面抗原，经乙肝抗原检测
试剂合检测，具有较强的抗体结合活性，且初步测定表达的抗原
蛋白最高占马铃薯可溶性总蛋白的0．34“
9、本试验虽然获得了预期结果，但对转基因马铃薯作为疫苗
的动物试验，以及遗传稳定性等试验等尚需时日，无法进行。虽
然我们同时进行了乙肝表面抗原基因HbsAg－s的转化试验，以检
验所设计的技术路线的有效性。但为了实现商品化的植物疫苗生
产目的，尚需进一步研究解决取材、方法、转化、筛选和检测等
一系
Potato(So]anuni tuberosum L) plants were firstly transformed with
the gene(h5nla) encoding an Avian Influena A (H5NI)Virus, and the
gene was expressed in transgenic potato. Meanwhile, the transfer
system was optimized in order to realize the aim of transgenic plant as
low-cost vaccine production system. The results were as follows.
1. To optimize the regeneration system of potato using tuber disc
as explants, it is necessary for the chosen media to have the high
efficient regeneration shoots of tuber disc. Using MS as the basic
medium and adjusting the hormone content and rate, four media were
obtained which were Ml, M2, M3, M4. By comparing the regeneration
rate of tuber discs on the four media, M4(MS-f-2.Omg/L BA+3.OmgIL
ZT+0.5mgIL NAA+0.Smg/L GA3) was selected as the highest efficient
one for shoots regeneration. And on M4 medium, ahead of time 20 days
or so, shoots could be regeneraed from 89.4 percent tuber discs.
Meanwhile care was taken to select more efficient media combination
for making plants stronger and producing more tubers. The media were
M7( I /2MS+0. 5mgIL BA+0. 5mg/L GA3+3%Suc) combinating with
M14(MS?.Omg/L BA+3.Omg/L ZT+l0%Suc) , by which the number
of tubers produced was twice as much as that on other media. The high
efficient regeneration system is essential for high efficient transforma-
tion system.
2. Four plant expression plasmids were constructed for
transformation. P13O1BG was constructed for expressing green
fluorescent protein gene(egfr); pl3OlB Q G was for enhanced
4
expression of eg4~, by inserting a 65bp enhancer( Q) between 35s
promoter and gene egfp; p13OIH5NI was for expression of Avian
Influena A (H5N1)Virus gene h5nla; pl3OlHBs was for expression a
small subunit protein of Hepatitis B Surface Antigen(HBsAg). In the
four plasmids, the CaMV35s promoter and nos terminator fragments
were syncretized with reporter gene and targeted gene. The selectable
maker gene terminated by polyA fragment for transformation of plants
was hygromycin phosphotransferase gene which allows selection with
hygromycin. And the selectable maker gene for plasmids was neomycin
phosphotransferae gene, which allows selection with kanamycin.
Agrobacterium strains LBA4404, EHAIO5 and AGL1 cells were
transformed by the direct method with plasmids mentioned above
prepared from Escherichia coil clones.
3. The transformation results mediated by gunpowder(ZHFQ-2),
demonstrated?that the explants were seriously damaged during the
transfer process, and no transfer plants were selected; By contrast, the
results mediated by Agrobacterium were stable.
4. By comparing several conditions of transformation mediated by
Agrobacterium, we found that the transfer efficiency was highly
effected by strains, content of strain cells, hormone, and AS etc. Among
the three strains (LBA4404, EH1O5 and AGL1), the invasiveness of the
strain EHA 105 was the highest. The GUS transient activity under
different transfer conditions showed that the following conditions
selected were much more efficient for transformation: Agrobacteria
that were resuspended and diluted with solution(MS+0.Smg/L
GA3+AS I Oumol/L) four times, invaded potato tuber discs swirring at
lOOrpmI28 0C for 20-30mm. Following co-cultivating, the explants
were transfered to M4 medium with the addition of 30 u mo]IL AS
表达，并优化了转化系统，为商品化生产转基因植物疫苗提供科
学依据。
1、为了优化马铃薯（Solanum tuberosum L.）以块茎为受体
的再生系统。着重对诱导分化和结薯培养基作了较系统的筛选。
以MS为基本培养基，调整激素配比，得到M1、M2、M3、M4等4
种培养基。比较试验结果以M4（MS＋2.0mg／L BA＋3.0mg／L ZT＋0.5mg／L
NAA＋0.5mg／L GA_3）诱导块茎分化的效果最佳，丛芽分化频率达89.4％，
分化时间提早约20天。同时筛选了诱导结薯的较优培养方法，获
得使试管薯结薯数增加近1倍的壮苗和诱导结薯培养基组合（壮
苗：1／2MS＋0.5mg／L BA＋0.5mg／L GA_3＋3％Suc；诱导结薯：MS＋2.0mg／L
BA＋3.0mg／L ZT＋10％Suc），为获得高频转化奠定了基础。
2、构建了4个植物表达双元载体。p1301BG含有增强型绿色
莹光蛋白基因egfp；p1301BΩG在egfp与35s启动子之间插入65bp
的增强子序列Ω因子；p1301H5N1含有禽流感病毒基因h5n1a；
p1301HBs含有乙肝表面抗原基因HBsAg。这4个载体均以35s为
真核表达启动子，报告基因和目的基因以Tnos为终止子、植物筛
选标记基因以35s PolyA加尾，融合成嵌合基因。原核抗性为Kan。
分别用直接法导入农杆菌EHA105、LBA4404和AGL1。
3、初步比较了ZHFQ-2型火药式基因枪与农杆菌的转化效果，
发现，该基因枪微弹分散性差，转化结果不稳定，对受体材料损
伤较大，未能筛选到转化株；而农杆菌转化相对较稳定，并初步
发现外源基因的瞬时表达大多集中在受伤和形成层部位。
4、对农杆菌转化条件的比较研究发现，菌株、菌体状态、稀
中文摘要
释度以及真空、激素和AS处理，均对转化有很大影响。对3个菌
株（LBA4404、EHA105和AGLI）的侵染性比较发现，用EHA105转化
马铃薯块茎，GUS瞬时表达效果更好；比较几种转化条件下的GUS
瞬时表达结果，筛选到可获得高且稳定的GUS瞬时表达的转化条
件：菌体用 Mg（MS＋0．sing／L GA3＋10umol／L AS）液体培养基稀释
4倍，与受体于28oC、100rpm振荡侵染20－30min，共培养时在分
化培养基上加 30卜 mol／L AS（乙酚丁香酮），16hr弱光照共培养 3d，
使GUS瞬时表达活性提高了3．4倍。
5、在进行抗生素基础抗性测定和转化体筛选时，比较马铃薯块茎
对Hgg和Kan两种筛选剂的敏感性，发现马铃薯块茎受体对Hnn更
敏感，因此选用Hgg为抗性筛选剂。在此基础上改进了筛选策略，
提出间段压力筛选法。该方法与常规的连续压力筛选法相比，大
大减低了基础抗性确定的主观性对转化效率的影响。使获得抗性
株的频率提高了82儿外源基因阳性率提高了2．4倍，获得转化株
的时间提早7－10天。
6、获得 HSNI转基因马铃薯株系 31个，并对其外源基因的整
合和表达做了系统检测。GUS表达活性结果表明，外源基因己插入
马铃薯基因组并得到表达，但株系间差异较大，说明外源基因整
合的随机性。用hsnla双引物扩增马铃薯基因组总DNA，得到与
目的基因大小一致的特异性片段。以DIG标记的hsnla探针进行
Southern杂交，进一步证实基因hsnla已整合到马铃薯基因组中，
且初步确定为单拷贝插入。
7、用HSNI 鼠单抗与马铃薯块茎蛋白粗提液进行抗原抗体反
应证实，在马铃薯中表达的外源基因具有抗原抗体反应活性，这
是禽流感病毒（HSNI）基因首次在植物中的表达。由于禽流感病
2
禽流感病毒（HSNI）基因（hjnla）在马铃薯中的转化与表达研究
毒m）是新病毒株，最近才得到其鼠的单克隆抗体，还末能
进行定量检测，以及动物免疫实验结果检测。
8、为了证实所建立的转化系统的有效性，本研究同时进行了
乙肝表面抗原＊BSAg）基因在马铃薯中的表达研究，并对结果做
了详细报道。在马铃薯中表达的乙肝表面抗原，经乙肝抗原检测
试剂合检测，具有较强的抗体结合活性，且初步测定表达的抗原
蛋白最高占马铃薯可溶性总蛋白的0．34“
9、本试验虽然获得了预期结果，但对转基因马铃薯作为疫苗
的动物试验，以及遗传稳定性等试验等尚需时日，无法进行。虽
然我们同时进行了乙肝表面抗原基因HbsAg－s的转化试验，以检
验所设计的技术路线的有效性。但为了实现商品化的植物疫苗生
产目的，尚需进一步研究解决取材、方法、转化、筛选和检测等
一系
Potato(So]anuni tuberosum L) plants were firstly transformed with
the gene(h5nla) encoding an Avian Influena A (H5NI)Virus, and the
gene was expressed in transgenic potato. Meanwhile, the transfer
system was optimized in order to realize the aim of transgenic plant as
low-cost vaccine production system. The results were as follows.
1. To optimize the regeneration system of potato using tuber disc
as explants, it is necessary for the chosen media to have the high
efficient regeneration shoots of tuber disc. Using MS as the basic
medium and adjusting the hormone content and rate, four media were
obtained which were Ml, M2, M3, M4. By comparing the regeneration
rate of tuber discs on the four media, M4(MS-f-2.Omg/L BA+3.OmgIL
ZT+0.5mgIL NAA+0.Smg/L GA3) was selected as the highest efficient
one for shoots regeneration. And on M4 medium, ahead of time 20 days
or so, shoots could be regeneraed from 89.4 percent tuber discs.
Meanwhile care was taken to select more efficient media combination
for making plants stronger and producing more tubers. The media were
M7( I /2MS+0. 5mgIL BA+0. 5mg/L GA3+3%Suc) combinating with
M14(MS?.Omg/L BA+3.Omg/L ZT+l0%Suc) , by which the number
of tubers produced was twice as much as that on other media. The high
efficient regeneration system is essential for high efficient transforma-
tion system.
2. Four plant expression plasmids were constructed for
transformation. P13O1BG was constructed for expressing green
fluorescent protein gene(egfr); pl3OlB Q G was for enhanced
4
expression of eg4~, by inserting a 65bp enhancer( Q) between 35s
promoter and gene egfp; p13OIH5NI was for expression of Avian
Influena A (H5N1)Virus gene h5nla; pl3OlHBs was for expression a
small subunit protein of Hepatitis B Surface Antigen(HBsAg). In the
four plasmids, the CaMV35s promoter and nos terminator fragments
were syncretized with reporter gene and targeted gene. The selectable
maker gene terminated by polyA fragment for transformation of plants
was hygromycin phosphotransferase gene which allows selection with
hygromycin. And the selectable maker gene for plasmids was neomycin
phosphotransferae gene, which allows selection with kanamycin.
Agrobacterium strains LBA4404, EHAIO5 and AGL1 cells were
transformed by the direct method with plasmids mentioned above
prepared from Escherichia coil clones.
3. The transformation results mediated by gunpowder(ZHFQ-2),
demonstrated?that the explants were seriously damaged during the
transfer process, and no transfer plants were selected; By contrast, the
results mediated by Agrobacterium were stable.
4. By comparing several conditions of transformation mediated by
Agrobacterium, we found that the transfer efficiency was highly
effected by strains, content of strain cells, hormone, and AS etc. Among
the three strains (LBA4404, EH1O5 and AGL1), the invasiveness of the
strain EHA 105 was the highest. The GUS transient activity under
different transfer conditions showed that the following conditions
selected were much more efficient for transformation: Agrobacteria
that were resuspended and diluted with solution(MS+0.Smg/L
GA3+AS I Oumol/L) four times, invaded potato tuber discs swirring at
lOOrpmI28 0C for 20-30mm. Following co-cultivating, the explants
were transfered to M4 medium with the addition of 30 u mo]IL AS
引文
1.Arakawa T, Chong DK, Slattery CW, et al, 1999. Improvements in human health through production of human milk proteins in transgenic food plants. Adv Exp Med Biol, 464:149-159.
2.Arakawa T,Chong DKX, Langridge WHR, et al, 1998. Efficency or a food plant-based oral cholera toxin B subunit vaccine, Nature Biotechnology 16(3):292-297.
3.Moffat A.S, 1995. Exploring transgenic plants as a new vaccine source,Science, 268(5):658-660.
4.Arakawa T, Chong DKX, Merritt JL, et al., 1997. Expression of cholera toxin B subunit oligomers in transgenic potato plants. Transgenic Res, 6(6):403-13.
5.Arakawa T, Yu J, Chong DKX, Hough J, et al, 1998. A plant-based cholera toxin B subunit-insulin fusion protein protects against the development of autoimmune diabetes, Nature Biotechnology vol(18):934-938.
6.Arntzen C J, Chong DKX, Chong DK, et al, 1992. Expression of hepatitis B surface antigene in transgenic plants. Proc Natl Acad Sci USA, 89:11745-11747.
7.Arntzen C.J, 1998. Immunity for Breakfast, Science, 280(8):p831.
8.Arntzen CJ & Langridge WH, 1998. Pharmaceutical foodstuffs oral immunization with transgenic plants, Nature Medicine, May supplement 4(5):p502-503.
9.Bender C, Hall H, Huang J, et al, 1999. Characterization of the surface proteins of influenza A (H5N1) viruses isolated from humans in 1997-1998.Virology, 254(1): 115-23.
10.Birch R G, 1997. Plant Transformation: Problem and Strategies for Practical Application, Annu.Rev. Plant Physiol. Plant Mol. Biol., 49: 297-326.
11.Zhou NN, Shortridge KF, Claas ECJ, et al, 1999. Rapid Evolution of H5N1 influenza viruses in chickens in Hong Kong, Journal of Virology, 73(4):3366-3374.
12.Block D & Herrera M, 1984. Expression of foreign genes in regenerated plants and their progeny, EMBO J.; 3: 1681-1689.
13.Bosch D, Smal J & Krebbers E, 1994. A tout growth hormone is expressed,correctly fold, and partially glycosylated in the leaves but not the seeds of transgenic plants, Transgene Res; 3(5): 304-310.
14.Buxton Bridges C & Krauss SL, 2000. Risk of influenza A (H5N1)infection among health care workers exposed to patients with influenza A (H5N1), Hong Kong. J Infect Dis; 34(2): 24-29.
15.Bower R, Elliott RA & Potier BAM, 1996. High-efficiency microprojectile mediated cotransformation of sugar cane, using visible or selectable markers.Mol.Breeding: 2: 239-249.
16.Chan M-T, Chang T-T & Ho S-L, 1993. Agrobacterium-mediated roduction of transgenic Rice plants expressing a chimerical α-amylase promoter/β-glucuronidase gene. Plant Mol. Biol.; 22: 491-506.
17.Chilton M.D, Drummond M.H & Merlo D.J, 1977. Stable incorporation of plasmid DNA into higher plant cells: the molecular basis of crown gall tumerigenesis. Cell; 11:262-271.
18.Chilton M-D, 1982. Integration and transcription of Ti plasmid fragments. P:299-319. In G.Kahl and J Schell, Molecular Bioclogy of Plant Tumors. Press:Inc., New York.
19.Chong DK, Roberts W, Arakawa T, et al., 1997. Expression of the human milk protein beta-casein in transgenic potato plants. Transgenic Res.; 6(4):289-296.
20.Christou P, 1995. Strategies for variety-independent genetic transformation of important cereals, legumes and woody species utilizing particlesbombardment, Euphytica; 85:13-27.
21.Claus S, Caroline S, & Michael W, 1998. The activities of acidic and transcriptional activation domains in plant cells: design of modular transcription factors for high-level expression, Plant Molecular Biology; 36:195-204.
22.Dale P.J.& Hampson K.K, 1995. An assessment of morphogenic and transformation efficency in a range of varietis of potato(Solanum tuberosum L.). Euphytica; 85:101-108.
23.Dale D.J, 1995. R and D regulation and field trailing of transgenic crops.Trends of Biotechnology; 13:398-403.
24.Dale PJ. & M.C. Mcpartlan. 1992. Field performance of transgenic potato plants compared with controls regenerated from tuber discs and shoot cuttings, Theor. Appl. Genet.; 84: 585-591.
25.David L. Suarez., 1998. Comparisons of highly virulent H5N1 influenza, A viruses isolated from humans and chickens from Hong Kong, Journal of virology; 72(8): 6678-6688.
26.De Block M, Herrera-Estrella L, Van Montage M, et al, 1984. Expression of foreign genes in regenerated plants and their progeny, EMBO J; 3:1681-89.
27.De Block M, De Sonville A, Debrouwer D, et al, 1995. The selection mechanism of phosphinothricin is influenced by the metabolic state of the tissue, Planta; 197:619-626.
28.De Jong J. C, Osterhaus M. E, Webster R. G, et al, 1997. A pandemic warning?, Nature; 389:554.
29.De loose M, Danthine X & Bockstaele E, 1995. Different 5' leader sequences modulated glucuronidase accumulation levels in transgenic Nicotiana tubacum. Plants Euphytica; 85:209-216.
30.Dong J, Teng W and Buchholz WG, 1996. Agrobacterium-mediated transformation of Javanica Rice, Molecular Breeding; 2:267-276.
31.Drummond, M.H, and Chilton M-D, 1978. Tumor inducing(Ti) plasmid of Agrobacterium share extensive regions of DNA homology, J. Bacteriol;136:1178-1183.
32.During K, Hippe S, Kreuzaler F, et al, 1995. Synthesis and self-assembly of a functional monoclonal antibody in transgenetic tobacco, Plant Mol. Biol.;15(2): 281-293.
33.Fies W, Riberdy J, Flynn K, et al, 1999. Protection against lnfluenze Virus in a mammalian system using M2HB, Nature Biotechnology; 22(9):856-862.
34.Gafni Y, Icht M, and Rabinfeld BZ, 1995. Stimulation of Agrobacterium tumefaciens virulence with indole-3-acetic acid. Lett. Appl. Microbiology;20: 98-101..
35.Gambley RI, Bryant JD and Masel NP, 1994. Cytokinin-enchanced regeneration of plants from microprojectile bombarded sugarcane meristematic tissue. Aust.J.Plant Physiol; 21:603-612.
36.Ganz PR, Dudani AK, Tackaberry ES, et al, 1996. In Transgenic Plant: A Production System for Industrial Proteins. Edited by Owen MR and Pan J.John Wiley and Sons Ltd.New York, NY. pp:281-297.
37.Geballe AP, Morris PJ, Lim W, et al, 1994. Initiation codon within 5'-leaders of mRNAs as regulators of translation, Trends Biochem. Sci.; 19:159-164.
38.Gil P, and GreGen PJ, 1996. Multiple regions of the Arabidopsis SAUR-ACI gene control transcript abundance: the 3'untranslated region functions as mRNA instability determination, EMBO J.; 15:1678-1686.
39.Goddijn O & Pen J, 1995. Plants as bioreactors, Trends in Biotechnology;13(9): 379-387.
40.Guyon P, Chilton M-D & Petit A, 1980. 'Agropine in null-type' crown gall tumors: evidence for generality of the opine concept, Proc Natl. Acad. SCI.,USA 77:2693-2697.
41.Haldrup A, Petersen SG, Okkels FT, et al, 1998. The xylose isomerase gene from Thermoanaerobacterium thermosul furogenes allows effective selection of transgenic plant cells using D-xylose as the selection agent,Plant Mol. Bio,; 37:287-296.
42.Hamamoto H, Sugiyama Y, Nakagawa N, et al, 1993. A new tobacco mosaic virus vector and its use for the systemic production of angiotensin-I-converting enzyme inhibitor in transgenic tobacco and tomato. Biotechnol;11(8): 930-932.
43.Haq TA, Mason HS, Arntzen CJ, et al, 1995. Oral immunization with a recombinant bacterial antigen produced in transgenic plants, Science May 5; 268(5211): 714-716.
44.Helen M., Lynn Tu., Godfrey W & Samuel S.M, 1998, Expression of the Brazil nut methionine-rich protein and mutants with increased methionine in transgenic potato, Plant molecular biology; 37:829-838.
45.Hensgens LAM & Clements JD, 1992. Translation controls the expression level of a chimeric reporter gene, Plant Mol. Biol., 20:921-938.
46.Herbers K, Sonnewald U, 1996. Manipulating metabolic partition in transgenic plants. Trends in Biotechnology; 14(6): 198-205.
47.Hiatt A, Cafferkey R., Bowdish B, et al, 1989. Production of antibodies in transgenetic plants. Nature; 342:76-78.
48.Higgins, E.S., Hlme J.S. & Shields R., 1992. Early events in transformation of potato by Agrobacierium tumefaciens, Plant Sci.; 82:109-118.
49.Higo KI, Saito Y, Higo H, et al, 1993. Expression of a chemically synthesized gene for human epidermal growth factor under the control of cauliflower mosaie virus 35s promoter in transgenic tobacco. Biosci Biotech Biochem; 57(9): 1477-1481.
50.Hockema A. & Dekker BMM, 1990. Production of correctly processed human serum albamin in transgenic plant-gene cloning and expression in tobacco and potato transgenic plant leaf and suspension cell culture: protein section, Journal of Cell
Biochemistry; 14(5): 333-335.
51.Hoekema A., Hirsch, P.J, and Schilperoort R.A, 1983. A binary plant vector strategy based on separation of vir-and T-region of the Agrobacterium tumefaciens(Ti) plasmid, Nature (London); 303:179-180.
52.Hohm B, Koukolikova-Nicha I, Bakkeren G et al, 1989. Agrobacteriummediated gene transfer to monocots and dicots, Genome; 31:987-991.
53.Hooykaas PJJ, Beijersbergen AGM, et al, 1994. The virulence system of Agrobacterium tume-faciens, Ann Rev of Phyto pathol; 32:157-179.
54.Horsch RB, 1984. Inheritance of functional foreign genes in plants. Science;223:496-498.
55.Horsch RB, Fraley RT and Rogers SG, 1984. Inheritance of functional foreign genes in plants. Science; 233:496-98.
56.Hwang Y.S, Karrer EE, and Thomas L, 1998. Three cis-elements required for rice α-amylase Amy3D expression during sugar starvation. Plant Molecular Biology; 36: 331-341.
57.Janice M. R, Kirsten J. F, Juergen S.R et al, 1999. Protection against a Lethal Avian Influenza A Virus in a Mammalian System, JVI; 73(2):1453-1459.
58.Julian K-L., Mich B., Hein H, et al, 1995. Immunotherapeutic potential of antibodies produced in plants, Tibtech December; 13(4): 522-526.
59.Ken-ichi H, Yasuhito S, Grimskey N, et al, 1993. Expression of chemically synthesized gene for human epidermal growth factor under the control of cauliflower mosaic virus 35s promoter transgenic tobacco. Bio Sci Biotech Biochem; 57: 1447-1481.
60.Komari T., 1990. Transformation of cultured cells of chenopodium quinoa by binary vectors that carry a fragment of DNA from the virulence region of pTiBo542. Plant Cell Rep. 9:303-306.
61.Laparra H, Burrus M, Hunold R, et al, 1995. Expression of foreign genes in sunflower: evaluation of three gene transfer methods, Euphytica; 85:63-74.
62.Leroux-Roels G & Grimskey N, 1997. Hepatitis B vaccine containing surface antigen and seleted proS1 and preS2 sequences, Vaccine;15(16):1724-1736.
63.Ma JK, Hiatt A, Hein M, et al, 1995. Generation and assembly of secretory antibodies in plants. Science; 268(5211): 716-719.
64.Ma JK, Lehner T, Stabila P, et al, 1994. Assembly of mono-clonal antibodies with IgG and IgA heavy chain domain in transgenic tobacco plants, Europe Journal of Immunology; 24(1): 131-138.
65.Maas C, Stvaric H, Escher A, et al, 1991. The combination of a noval stimulatory element in the fist exon of the maize shranken-1 gene with the following intron 1 enhances reporter gene expression up to 1000 fold. Plant Mol. Biol.; 16: 199-207.
66.Mason HS, Ball JM, Shi JJ, et al, 1996. Expression of Norwalk virus capsid protein in transgenic tobacco and potato and its oral immunogenicity in mice,Proc Natl Acad Sci USA; 93(11): 5335-5340.
67.Mason HS, Lam DM, Arntzen CJ, et al, 1992, Expression of hepatitis B antigen in transgenic plants, Proc Natl Acad Sci USA; 89(24):11745-11749.
68.Mason HS & Arntzen CJ, 1995. Transgenic plants as vaccine production systems, Trends in Biotechnology; 13(9): 388-392.
69.May GD, Arntzen CJ, Mason HS, et al, 1995. Generation of transgenic banana(Musa acuminata) plants via Agro-bacterium-mediated transformation,Biotechnology; 13(5): 486-492.
70.Michael F, Sun J-B, Yang YM, et al, 1997. A promoter directing high expressing in pistils of transgenic plants, PLant Molecular Biology; 35:425-431.
71.Michael WG, Stuart C and Peter MW, 1997. Expression patterns of vascularspecific promoters RolC and Sh in transgenic potatoes and their use in engineering PLRV-resistant plants, Plant Molecular Biology; 33:729-735.
72.Miele L, 1997, Plants as bioreactors for biophar-maceuticals: regulatory consideration, TIBTECT; 15(2):45-50.
73.Mitsuhara I, Ugaki M, and Hirochika H, 1996. Efficienty promoter cassettes for enhanced expression of foreign genes in dicotyledonous and monocotyledonous plants Plants Cell Physiol; 37(1): 49-59.
74.Moffat AS, 1995. Exploring Transgenic Plants As a New Vaccine Source.Scince; 268:658-660.
75.Nathan S., & Irwin J., 1998. Lectins: More than Insecticides, Science;281(5380): 1124-1125.
76.Naturforsch Z, Mason HS, Elson CO,et al, 1998. Transgenic potato plants expressing soybean beta-1,3-endoglucanase gene exhibit an increased resistance to Phytophthora infestans, Plant Molecular Biology; 53(11-12):1012-1016.
77.Newell C.A, Rozman R, Hinchee M.A, et al, 1991. Agrobacierium mediated transformation of Solanum Tuberosum L. Cv.Russet Burbank,Plant Cell Rep; 10:30-34.
78.Owen M, Gandecha A, Cockburn B, et al, 1992. Synthesis of a functional anti-phytochrome single chain Fv protein in transgenic tobacco, Nature Biotechnology; 10(7): 790-794.
79.Parmenter DL, Boothe JG, Van Rooijer GJH, et al, 1995. Pro-duction of biologically active hirudin in plant seed using oleosiu partitoning, Plant Mol Biol; 29(6): 1167-1180.
80.Pasikowski J, Shillito RD, Saul M, et al, 1984. Direct gene transfer to plants.EMBO J.; 3: 2717-2722.
81.Paul MH and John AG, 1997. A 38bp repeat sequence within the pea seed storage protein promoter of legA is a binding site for a nuclear DNA-binding protein, Plant Molecular Biology; 33:175-180.
82.Petre J, Ganem D, Cabral S, et al, 1992. Properties of a recombinant yeastderived hepatitis B surface antigen containing S, preS2 and preS1 antigenic domains, Arch. Virol; 4:137-141.
83.Pilip J. Dale & Kaija K. Hampson, 1995. An assessment of morphogenic and transformation efficiency in arrange of varieties of potato(Solanum tuerosum L.)Euphytica; 5:101-108.
84.Poklemba C.J., & Fjellstrom R.G, 1995. A rapid one-tuber genomic DNA extraction process for PCR and RAPD analyses, Nucleic Acids Research;23(13): 2569-2570.
85.Pride M, Cabral S, Hinchee M.A, et al, 1998. Evaluation of B and T-cell responses in chimpanzees immunized with Hepagene, apepatitis B vaccine containing pre-S1/preS2 gene products. Vaccine; 16: 543-550.
86.Prufer D., Schmitz J., Tacke E, et al, 1997. A full-length eDNA copy(PLRVF1)of potato leafroll virus(PLRV) in protoplasts and transgenic plants,Mol. Gen. Genet; 253(5):609-614.
87.Rainier DM, Bottino P and Gordon MP, 1993. Agrobacterium-mediated transformation of rice (Oryza Sativa L.). Bio/technology 8:33-38.
88.Richardson JH & Marasco WA, 1995. Intracellular antibodies: development and therapeutic potential, Trends in Biotechnology; 13(8): 306-309.
89.Russell DR, Wallace KM, Bathe JH, et al, 1993. Stable transformation of Phaseolus vulgaris via electric-discharge mediated particle acceleration,Plant Cell Rep; 12:165-169.
90.Sanchayita K, Kaija K, Hampson W, et al, 1996. Efficient transgenic plant regeneration through Agrobacterium-mediated trans-formation of CHICKPEA, Plant Cell Report; 16:32-37.
91.Schouten A, Cockburn B, Schrammeijer B, et al, 1997. Improving scFv antibody expression levels in the plant cytosol. FEBS Letter; 415(2): 235-41.
92.Sheeman,S. & M.W. Bevan, 1988. A rapid transformation method for Solanum tuerosum using binary Agrobacierium tumefaciens vector. Plant Cell Rep.; 7:13-16.
93.Shimamoto K, Terada R, and Izawa T., 1989. Fertile tran-sgenic rice plants regenerated from transformed protoplasts, Nature; 338:274-276.
94.Siela N.M, Abhaya M.D, and Mark JG., 1998. Investigation of Agrobacterium-mediated transformation of apple using green fluorescent protein: high transient expression and low stable transformation that factors other than T-DNA transfer are rate-limiting, Plant Mol. Biology; 37:549-559.
95.Sijmons PC, Dekker BMM, Schrammeijer B, et al. 1990. Production of correctly processed human serum albumin in transgenic plants, Nature Biotechnology; 8(3): 217-221.
96.Smith E.F., 1909. The etiology of plant tumors, Science; 30: 223.
97.Smith MD, Dekker BMM, Cockburn B, et al, 1996. Antibody production in plants. Biotechnology Advance, 14(3):267-281.
98.Songstad DD & Somer DA, 1995. Advances in alternative DNA delivery techniques, Plant Cell Tissue and Organ Culture; 40(1). 90-95.
99.Sonke H, Jutta L-M; Klans A, et al, 1998. High-level expression of a viscotoxin in Arabidopsis thaliana gives enhanced resistance against Plasmodiophora brassicae, PMB; 36: 673-680.
100.Spiker S, & Thompson WF, 1996. Nuclear matrix attanchment regions and transgene expression in plants, Plant Physiol; 110:15-21.
101.Stiekema WJ, Heidekamp F, Louwerse JD et al, 1988. Introduction of foreign genes into potato cultivars Bintje and Desiree using an Agrobacterium tumefaciens binary vector, Plant Cell Rep; 7:47-50.
102.Stockhaus J, Jefferson RA, Karp A, et al, 1987. Organ-specific and dosagedependent expression of a leaf/stem specific gene from potato after tagging and transfer into potato and tobacco plants. Nucleic Acids Res; 15(8): 3479-91.
103.Suarez D L., Perdue M L., Swayne D E, et al. 1998. Comparisons of Highly Virulent H5N1 influenza A Viruses Isolated from Humans and Chickens from Hong Kong, Journal of Virology; 72(8):6678-6688.
104.Subbarao K, Klimov A, Katz J, et al, 1998. Characterization of an Avian Influenza A(H5N1) Virus isolated from a child with a fatal respiratory illness,Science 279; 16(5349):p393-396.
105.Tacket C.O, Mason H.C, Losonsky G, & Arntzen C.J, 1998. Immunogenicity in humans of a recombinant bacterial antigen delivered in a transgenic potato,Nature Medicine; (14)5: p607.
106.Takase K, Hagiwara K, Schrammeijer B, et al, 1998. Expression of human alpha-lactalbumin in transgenic tobacco, J. Biochem; 123(3):440-444.
107.Tanbenberger, J.K., Gao P, Goto W, et al, 1998. Infiuenza virus hemayglutinin cleavage into Hal. Haz: No lauyhing matter, Proc. Natl. Acid.Sci.USA; 95:9713-9715.
108.Tavladoraki P, Benvenuto T, Losonsky G, et al, 1993. Transgenic plants expression a functional single-chain Fv antibody specifically protected from virus attack, Nature; 366: 469-472.
109.Thanavala Y, Arntzen C J, Schrammeijer B, et al, 1995. Immunogenicity of transgenic plant-derived hepatitis B surface antigen, Proc Natl Acad Sci USA; 92:3358-3361.
110.Van Engelen FA, Schouten A, Molthoff JW, et al, 1994. Coordinate expression of antibody subunit genes yields high levels of functional antibodies in roote of transgrnic tobacco, Plant Mol Biol; 26(6): 1701-1710.
111.Van Rooijen GJH & Moloney MM, 1998. Plant seed oil-bodies as carriers for foreign proteins; Biotechnology; 13(1): 72-77.
112.Vander Wilk F, Posthumus D, Husiman MJ, et al, 1991. Expression of the potato leafroll luteovirus cost protein gene in transgenic potato plants inhibits viral infection, Plant Mol Biol; 17(3): 432-439.
113.Visser RG, Jacobsen E, Hasseling-M A, et al, 1989. Transformantion of homozygous diploid potato with an Agrobacterium tumefaciens binary vector system by adventitious shoot regeneration on leaf and stem segments,Plant Mol. Biol.; 12:329-337.
114.Wallis GJ, Dale PJ, Lander D, et al, 1997. Expression of a synthetic antifreeze protein in potato reduces electrolyte release at freezing temperatures. Plant Mol Biol.1997, 35(3):323-30.
115.Wan Y, Lemaux PG, Losonsky G, et al, 1994. Generation of large number of independently transformed fertile barley plants, Plant Physiol.; 104(5):37-48.
116.Wenzler H, Mignery G, May G, et al, 1989. A Rapid and efficient transformation method for the production of large numbers of transgenic potato plants, Plant Sci.; 63: 79-85.
88
117.Whitelam GC, Cockbum B, Verhoeven HA, et al, 1993、Heterologous protein production in transgenic plants, Biotechnology and Genetic Engineering Reviews; 11:1-29.
118.Wilde C, Neven M, Wilde C, et al, 1996. Intact antigen binding MAK33 antibody and fab fragment accumulate in intercellular space of Arabidopsis thaliana, Plant Sci.; 114(2):133-141.
119.Zaenen I Vanlarebeke N, 1974. Supercoiled circular DNA in crown-gall inducing agrobactrium strains, J. Mol Biol.; 86:109-127.
120.Zambryski P & Boston RS, 1992. Chronicles from the Agrobacterium-plant cells DNA transfer story, Ann Rev Plant Physiol; 43: 465-490.
121.Zuo WN, Weissinger AK, Boston RS, et al, 1995. Expression of a maize ribosomeinactivating protein gene in transgenic tobacco plants, Plant Physial Suppl.; 108(2): 150-152.
122.Kodihalli S, Goto H, Darwyn L.K, et al, 1999. DNA Vaccine Encoding Hemagglutinin Provides Protective Immunity against H5N1 Influenza Virus Infection in Mice, Journal of Virology; 73(3):2094-2098.
123．于静娟、敖光明、陈章良等，1997。水稻10KD富硫醇溶蛋白基因在马铃薯中的表达，植物学报；39(4)：329-334。
124．催武、刘炜、吴光耀等，1995。高效、快速地将外源DNA导入根癌土壤杆菌，生物工程学报；11(4)：350—355。
125．刘信、李宁1999。农业生物技术发展与基因工程的安全管理，农业科技管理；99(2)：7～9。
126．刘昕、傅荣昭、蔡民等，1998。SAR与植物转基因沉默的消除，生物工程进展；18(4)：53—58。
127．刘玉乐、田波，1995。 人乙肝病毒表面抗原基因在转基因烟草中的表达，中国科学(B辑)；23：252—255。
128．朱常香、潘春欣，1996。利用PIG基因枪进行水稻转化的研究，山东农业大学学报；27(2)：124—128。
129．朱桢、敖光明、陈章良等，1992。转基因水稻植株再生及外源人α-干扰素cDNA的表达，中国科学(B辑)：22：149-155。
130．王学、王海涛，1996。抗体库的发展及未来，生物化学与生物物理进展；23(4)：312～316。
131．王关林、方宏筠，1998。《植株基因工程原理与技术》，科学出版社；p：46—49。
132．贾士荣、曹冬孙，1992。转基因植物，植物学通报：9(2)：3—15。
133．赵倩、敖光明、于静娟等，1995。牛生长激素基因在马铃薯中的表达，植物学报；37(11)：840-847。
134．雷茂良、程金根，1998。全球转基因植物发展现状，生物技术通报：98 (6)：30～32。
135．刘涤，1999。转基因植物-生产药物的新型反应器，生物技术通报；3：23-27。
136．王新国、肖成祖，1998。用转基因植物生产基因工程疫苗，生物工程进展；18(1)：51-54。
137．顾红雅，翟礼嘉主译，(1998)《植物分子生物学-实验手册》高等教育出版社，北京。
138．金冬雁，黎孟枫等译(1992)《分子克隆》(第二版)，科学出版社，北京。
2.Arakawa T,Chong DKX, Langridge WHR, et al, 1998. Efficency or a food plant-based oral cholera toxin B subunit vaccine, Nature Biotechnology 16(3):292-297.
3.Moffat A.S, 1995. Exploring transgenic plants as a new vaccine source,Science, 268(5):658-660.
4.Arakawa T, Chong DKX, Merritt JL, et al., 1997. Expression of cholera toxin B subunit oligomers in transgenic potato plants. Transgenic Res, 6(6):403-13.
5.Arakawa T, Yu J, Chong DKX, Hough J, et al, 1998. A plant-based cholera toxin B subunit-insulin fusion protein protects against the development of autoimmune diabetes, Nature Biotechnology vol(18):934-938.
6.Arntzen C J, Chong DKX, Chong DK, et al, 1992. Expression of hepatitis B surface antigene in transgenic plants. Proc Natl Acad Sci USA, 89:11745-11747.
7.Arntzen C.J, 1998. Immunity for Breakfast, Science, 280(8):p831.
8.Arntzen CJ & Langridge WH, 1998. Pharmaceutical foodstuffs oral immunization with transgenic plants, Nature Medicine, May supplement 4(5):p502-503.
9.Bender C, Hall H, Huang J, et al, 1999. Characterization of the surface proteins of influenza A (H5N1) viruses isolated from humans in 1997-1998.Virology, 254(1): 115-23.
10.Birch R G, 1997. Plant Transformation: Problem and Strategies for Practical Application, Annu.Rev. Plant Physiol. Plant Mol. Biol., 49: 297-326.
11.Zhou NN, Shortridge KF, Claas ECJ, et al, 1999. Rapid Evolution of H5N1 influenza viruses in chickens in Hong Kong, Journal of Virology, 73(4):3366-3374.
12.Block D & Herrera M, 1984. Expression of foreign genes in regenerated plants and their progeny, EMBO J.; 3: 1681-1689.
13.Bosch D, Smal J & Krebbers E, 1994. A tout growth hormone is expressed,correctly fold, and partially glycosylated in the leaves but not the seeds of transgenic plants, Transgene Res; 3(5): 304-310.
14.Buxton Bridges C & Krauss SL, 2000. Risk of influenza A (H5N1)infection among health care workers exposed to patients with influenza A (H5N1), Hong Kong. J Infect Dis; 34(2): 24-29.
15.Bower R, Elliott RA & Potier BAM, 1996. High-efficiency microprojectile mediated cotransformation of sugar cane, using visible or selectable markers.Mol.Breeding: 2: 239-249.
16.Chan M-T, Chang T-T & Ho S-L, 1993. Agrobacterium-mediated roduction of transgenic Rice plants expressing a chimerical α-amylase promoter/β-glucuronidase gene. Plant Mol. Biol.; 22: 491-506.
17.Chilton M.D, Drummond M.H & Merlo D.J, 1977. Stable incorporation of plasmid DNA into higher plant cells: the molecular basis of crown gall tumerigenesis. Cell; 11:262-271.
18.Chilton M-D, 1982. Integration and transcription of Ti plasmid fragments. P:299-319. In G.Kahl and J Schell, Molecular Bioclogy of Plant Tumors. Press:Inc., New York.
19.Chong DK, Roberts W, Arakawa T, et al., 1997. Expression of the human milk protein beta-casein in transgenic potato plants. Transgenic Res.; 6(4):289-296.
20.Christou P, 1995. Strategies for variety-independent genetic transformation of important cereals, legumes and woody species utilizing particlesbombardment, Euphytica; 85:13-27.
21.Claus S, Caroline S, & Michael W, 1998. The activities of acidic and transcriptional activation domains in plant cells: design of modular transcription factors for high-level expression, Plant Molecular Biology; 36:195-204.
22.Dale P.J.& Hampson K.K, 1995. An assessment of morphogenic and transformation efficency in a range of varietis of potato(Solanum tuberosum L.). Euphytica; 85:101-108.
23.Dale D.J, 1995. R and D regulation and field trailing of transgenic crops.Trends of Biotechnology; 13:398-403.
24.Dale PJ. & M.C. Mcpartlan. 1992. Field performance of transgenic potato plants compared with controls regenerated from tuber discs and shoot cuttings, Theor. Appl. Genet.; 84: 585-591.
25.David L. Suarez., 1998. Comparisons of highly virulent H5N1 influenza, A viruses isolated from humans and chickens from Hong Kong, Journal of virology; 72(8): 6678-6688.
26.De Block M, Herrera-Estrella L, Van Montage M, et al, 1984. Expression of foreign genes in regenerated plants and their progeny, EMBO J; 3:1681-89.
27.De Block M, De Sonville A, Debrouwer D, et al, 1995. The selection mechanism of phosphinothricin is influenced by the metabolic state of the tissue, Planta; 197:619-626.
28.De Jong J. C, Osterhaus M. E, Webster R. G, et al, 1997. A pandemic warning?, Nature; 389:554.
29.De loose M, Danthine X & Bockstaele E, 1995. Different 5' leader sequences modulated glucuronidase accumulation levels in transgenic Nicotiana tubacum. Plants Euphytica; 85:209-216.
30.Dong J, Teng W and Buchholz WG, 1996. Agrobacterium-mediated transformation of Javanica Rice, Molecular Breeding; 2:267-276.
31.Drummond, M.H, and Chilton M-D, 1978. Tumor inducing(Ti) plasmid of Agrobacterium share extensive regions of DNA homology, J. Bacteriol;136:1178-1183.
32.During K, Hippe S, Kreuzaler F, et al, 1995. Synthesis and self-assembly of a functional monoclonal antibody in transgenetic tobacco, Plant Mol. Biol.;15(2): 281-293.
33.Fies W, Riberdy J, Flynn K, et al, 1999. Protection against lnfluenze Virus in a mammalian system using M2HB, Nature Biotechnology; 22(9):856-862.
34.Gafni Y, Icht M, and Rabinfeld BZ, 1995. Stimulation of Agrobacterium tumefaciens virulence with indole-3-acetic acid. Lett. Appl. Microbiology;20: 98-101..
35.Gambley RI, Bryant JD and Masel NP, 1994. Cytokinin-enchanced regeneration of plants from microprojectile bombarded sugarcane meristematic tissue. Aust.J.Plant Physiol; 21:603-612.
36.Ganz PR, Dudani AK, Tackaberry ES, et al, 1996. In Transgenic Plant: A Production System for Industrial Proteins. Edited by Owen MR and Pan J.John Wiley and Sons Ltd.New York, NY. pp:281-297.
37.Geballe AP, Morris PJ, Lim W, et al, 1994. Initiation codon within 5'-leaders of mRNAs as regulators of translation, Trends Biochem. Sci.; 19:159-164.
38.Gil P, and GreGen PJ, 1996. Multiple regions of the Arabidopsis SAUR-ACI gene control transcript abundance: the 3'untranslated region functions as mRNA instability determination, EMBO J.; 15:1678-1686.
39.Goddijn O & Pen J, 1995. Plants as bioreactors, Trends in Biotechnology;13(9): 379-387.
40.Guyon P, Chilton M-D & Petit A, 1980. 'Agropine in null-type' crown gall tumors: evidence for generality of the opine concept, Proc Natl. Acad. SCI.,USA 77:2693-2697.
41.Haldrup A, Petersen SG, Okkels FT, et al, 1998. The xylose isomerase gene from Thermoanaerobacterium thermosul furogenes allows effective selection of transgenic plant cells using D-xylose as the selection agent,Plant Mol. Bio,; 37:287-296.
42.Hamamoto H, Sugiyama Y, Nakagawa N, et al, 1993. A new tobacco mosaic virus vector and its use for the systemic production of angiotensin-I-converting enzyme inhibitor in transgenic tobacco and tomato. Biotechnol;11(8): 930-932.
43.Haq TA, Mason HS, Arntzen CJ, et al, 1995. Oral immunization with a recombinant bacterial antigen produced in transgenic plants, Science May 5; 268(5211): 714-716.
44.Helen M., Lynn Tu., Godfrey W & Samuel S.M, 1998, Expression of the Brazil nut methionine-rich protein and mutants with increased methionine in transgenic potato, Plant molecular biology; 37:829-838.
45.Hensgens LAM & Clements JD, 1992. Translation controls the expression level of a chimeric reporter gene, Plant Mol. Biol., 20:921-938.
46.Herbers K, Sonnewald U, 1996. Manipulating metabolic partition in transgenic plants. Trends in Biotechnology; 14(6): 198-205.
47.Hiatt A, Cafferkey R., Bowdish B, et al, 1989. Production of antibodies in transgenetic plants. Nature; 342:76-78.
48.Higgins, E.S., Hlme J.S. & Shields R., 1992. Early events in transformation of potato by Agrobacierium tumefaciens, Plant Sci.; 82:109-118.
49.Higo KI, Saito Y, Higo H, et al, 1993. Expression of a chemically synthesized gene for human epidermal growth factor under the control of cauliflower mosaie virus 35s promoter in transgenic tobacco. Biosci Biotech Biochem; 57(9): 1477-1481.
50.Hockema A. & Dekker BMM, 1990. Production of correctly processed human serum albamin in transgenic plant-gene cloning and expression in tobacco and potato transgenic plant leaf and suspension cell culture: protein section, Journal of Cell
Biochemistry; 14(5): 333-335.
51.Hoekema A., Hirsch, P.J, and Schilperoort R.A, 1983. A binary plant vector strategy based on separation of vir-and T-region of the Agrobacterium tumefaciens(Ti) plasmid, Nature (London); 303:179-180.
52.Hohm B, Koukolikova-Nicha I, Bakkeren G et al, 1989. Agrobacteriummediated gene transfer to monocots and dicots, Genome; 31:987-991.
53.Hooykaas PJJ, Beijersbergen AGM, et al, 1994. The virulence system of Agrobacterium tume-faciens, Ann Rev of Phyto pathol; 32:157-179.
54.Horsch RB, 1984. Inheritance of functional foreign genes in plants. Science;223:496-498.
55.Horsch RB, Fraley RT and Rogers SG, 1984. Inheritance of functional foreign genes in plants. Science; 233:496-98.
56.Hwang Y.S, Karrer EE, and Thomas L, 1998. Three cis-elements required for rice α-amylase Amy3D expression during sugar starvation. Plant Molecular Biology; 36: 331-341.
57.Janice M. R, Kirsten J. F, Juergen S.R et al, 1999. Protection against a Lethal Avian Influenza A Virus in a Mammalian System, JVI; 73(2):1453-1459.
58.Julian K-L., Mich B., Hein H, et al, 1995. Immunotherapeutic potential of antibodies produced in plants, Tibtech December; 13(4): 522-526.
59.Ken-ichi H, Yasuhito S, Grimskey N, et al, 1993. Expression of chemically synthesized gene for human epidermal growth factor under the control of cauliflower mosaic virus 35s promoter transgenic tobacco. Bio Sci Biotech Biochem; 57: 1447-1481.
60.Komari T., 1990. Transformation of cultured cells of chenopodium quinoa by binary vectors that carry a fragment of DNA from the virulence region of pTiBo542. Plant Cell Rep. 9:303-306.
61.Laparra H, Burrus M, Hunold R, et al, 1995. Expression of foreign genes in sunflower: evaluation of three gene transfer methods, Euphytica; 85:63-74.
62.Leroux-Roels G & Grimskey N, 1997. Hepatitis B vaccine containing surface antigen and seleted proS1 and preS2 sequences, Vaccine;15(16):1724-1736.
63.Ma JK, Hiatt A, Hein M, et al, 1995. Generation and assembly of secretory antibodies in plants. Science; 268(5211): 716-719.
64.Ma JK, Lehner T, Stabila P, et al, 1994. Assembly of mono-clonal antibodies with IgG and IgA heavy chain domain in transgenic tobacco plants, Europe Journal of Immunology; 24(1): 131-138.
65.Maas C, Stvaric H, Escher A, et al, 1991. The combination of a noval stimulatory element in the fist exon of the maize shranken-1 gene with the following intron 1 enhances reporter gene expression up to 1000 fold. Plant Mol. Biol.; 16: 199-207.
66.Mason HS, Ball JM, Shi JJ, et al, 1996. Expression of Norwalk virus capsid protein in transgenic tobacco and potato and its oral immunogenicity in mice,Proc Natl Acad Sci USA; 93(11): 5335-5340.
67.Mason HS, Lam DM, Arntzen CJ, et al, 1992, Expression of hepatitis B antigen in transgenic plants, Proc Natl Acad Sci USA; 89(24):11745-11749.
68.Mason HS & Arntzen CJ, 1995. Transgenic plants as vaccine production systems, Trends in Biotechnology; 13(9): 388-392.
69.May GD, Arntzen CJ, Mason HS, et al, 1995. Generation of transgenic banana(Musa acuminata) plants via Agro-bacterium-mediated transformation,Biotechnology; 13(5): 486-492.
70.Michael F, Sun J-B, Yang YM, et al, 1997. A promoter directing high expressing in pistils of transgenic plants, PLant Molecular Biology; 35:425-431.
71.Michael WG, Stuart C and Peter MW, 1997. Expression patterns of vascularspecific promoters RolC and Sh in transgenic potatoes and their use in engineering PLRV-resistant plants, Plant Molecular Biology; 33:729-735.
72.Miele L, 1997, Plants as bioreactors for biophar-maceuticals: regulatory consideration, TIBTECT; 15(2):45-50.
73.Mitsuhara I, Ugaki M, and Hirochika H, 1996. Efficienty promoter cassettes for enhanced expression of foreign genes in dicotyledonous and monocotyledonous plants Plants Cell Physiol; 37(1): 49-59.
74.Moffat AS, 1995. Exploring Transgenic Plants As a New Vaccine Source.Scince; 268:658-660.
75.Nathan S., & Irwin J., 1998. Lectins: More than Insecticides, Science;281(5380): 1124-1125.
76.Naturforsch Z, Mason HS, Elson CO,et al, 1998. Transgenic potato plants expressing soybean beta-1,3-endoglucanase gene exhibit an increased resistance to Phytophthora infestans, Plant Molecular Biology; 53(11-12):1012-1016.
77.Newell C.A, Rozman R, Hinchee M.A, et al, 1991. Agrobacierium mediated transformation of Solanum Tuberosum L. Cv.Russet Burbank,Plant Cell Rep; 10:30-34.
78.Owen M, Gandecha A, Cockburn B, et al, 1992. Synthesis of a functional anti-phytochrome single chain Fv protein in transgenic tobacco, Nature Biotechnology; 10(7): 790-794.
79.Parmenter DL, Boothe JG, Van Rooijer GJH, et al, 1995. Pro-duction of biologically active hirudin in plant seed using oleosiu partitoning, Plant Mol Biol; 29(6): 1167-1180.
80.Pasikowski J, Shillito RD, Saul M, et al, 1984. Direct gene transfer to plants.EMBO J.; 3: 2717-2722.
81.Paul MH and John AG, 1997. A 38bp repeat sequence within the pea seed storage protein promoter of legA is a binding site for a nuclear DNA-binding protein, Plant Molecular Biology; 33:175-180.
82.Petre J, Ganem D, Cabral S, et al, 1992. Properties of a recombinant yeastderived hepatitis B surface antigen containing S, preS2 and preS1 antigenic domains, Arch. Virol; 4:137-141.
83.Pilip J. Dale & Kaija K. Hampson, 1995. An assessment of morphogenic and transformation efficiency in arrange of varieties of potato(Solanum tuerosum L.)Euphytica; 5:101-108.
84.Poklemba C.J., & Fjellstrom R.G, 1995. A rapid one-tuber genomic DNA extraction process for PCR and RAPD analyses, Nucleic Acids Research;23(13): 2569-2570.
85.Pride M, Cabral S, Hinchee M.A, et al, 1998. Evaluation of B and T-cell responses in chimpanzees immunized with Hepagene, apepatitis B vaccine containing pre-S1/preS2 gene products. Vaccine; 16: 543-550.
86.Prufer D., Schmitz J., Tacke E, et al, 1997. A full-length eDNA copy(PLRVF1)of potato leafroll virus(PLRV) in protoplasts and transgenic plants,Mol. Gen. Genet; 253(5):609-614.
87.Rainier DM, Bottino P and Gordon MP, 1993. Agrobacterium-mediated transformation of rice (Oryza Sativa L.). Bio/technology 8:33-38.
88.Richardson JH & Marasco WA, 1995. Intracellular antibodies: development and therapeutic potential, Trends in Biotechnology; 13(8): 306-309.
89.Russell DR, Wallace KM, Bathe JH, et al, 1993. Stable transformation of Phaseolus vulgaris via electric-discharge mediated particle acceleration,Plant Cell Rep; 12:165-169.
90.Sanchayita K, Kaija K, Hampson W, et al, 1996. Efficient transgenic plant regeneration through Agrobacterium-mediated trans-formation of CHICKPEA, Plant Cell Report; 16:32-37.
91.Schouten A, Cockburn B, Schrammeijer B, et al, 1997. Improving scFv antibody expression levels in the plant cytosol. FEBS Letter; 415(2): 235-41.
92.Sheeman,S. & M.W. Bevan, 1988. A rapid transformation method for Solanum tuerosum using binary Agrobacierium tumefaciens vector. Plant Cell Rep.; 7:13-16.
93.Shimamoto K, Terada R, and Izawa T., 1989. Fertile tran-sgenic rice plants regenerated from transformed protoplasts, Nature; 338:274-276.
94.Siela N.M, Abhaya M.D, and Mark JG., 1998. Investigation of Agrobacterium-mediated transformation of apple using green fluorescent protein: high transient expression and low stable transformation that factors other than T-DNA transfer are rate-limiting, Plant Mol. Biology; 37:549-559.
95.Sijmons PC, Dekker BMM, Schrammeijer B, et al. 1990. Production of correctly processed human serum albumin in transgenic plants, Nature Biotechnology; 8(3): 217-221.
96.Smith E.F., 1909. The etiology of plant tumors, Science; 30: 223.
97.Smith MD, Dekker BMM, Cockburn B, et al, 1996. Antibody production in plants. Biotechnology Advance, 14(3):267-281.
98.Songstad DD & Somer DA, 1995. Advances in alternative DNA delivery techniques, Plant Cell Tissue and Organ Culture; 40(1). 90-95.
99.Sonke H, Jutta L-M; Klans A, et al, 1998. High-level expression of a viscotoxin in Arabidopsis thaliana gives enhanced resistance against Plasmodiophora brassicae, PMB; 36: 673-680.
100.Spiker S, & Thompson WF, 1996. Nuclear matrix attanchment regions and transgene expression in plants, Plant Physiol; 110:15-21.
101.Stiekema WJ, Heidekamp F, Louwerse JD et al, 1988. Introduction of foreign genes into potato cultivars Bintje and Desiree using an Agrobacterium tumefaciens binary vector, Plant Cell Rep; 7:47-50.
102.Stockhaus J, Jefferson RA, Karp A, et al, 1987. Organ-specific and dosagedependent expression of a leaf/stem specific gene from potato after tagging and transfer into potato and tobacco plants. Nucleic Acids Res; 15(8): 3479-91.
103.Suarez D L., Perdue M L., Swayne D E, et al. 1998. Comparisons of Highly Virulent H5N1 influenza A Viruses Isolated from Humans and Chickens from Hong Kong, Journal of Virology; 72(8):6678-6688.
104.Subbarao K, Klimov A, Katz J, et al, 1998. Characterization of an Avian Influenza A(H5N1) Virus isolated from a child with a fatal respiratory illness,Science 279; 16(5349):p393-396.
105.Tacket C.O, Mason H.C, Losonsky G, & Arntzen C.J, 1998. Immunogenicity in humans of a recombinant bacterial antigen delivered in a transgenic potato,Nature Medicine; (14)5: p607.
106.Takase K, Hagiwara K, Schrammeijer B, et al, 1998. Expression of human alpha-lactalbumin in transgenic tobacco, J. Biochem; 123(3):440-444.
107.Tanbenberger, J.K., Gao P, Goto W, et al, 1998. Infiuenza virus hemayglutinin cleavage into Hal. Haz: No lauyhing matter, Proc. Natl. Acid.Sci.USA; 95:9713-9715.
108.Tavladoraki P, Benvenuto T, Losonsky G, et al, 1993. Transgenic plants expression a functional single-chain Fv antibody specifically protected from virus attack, Nature; 366: 469-472.
109.Thanavala Y, Arntzen C J, Schrammeijer B, et al, 1995. Immunogenicity of transgenic plant-derived hepatitis B surface antigen, Proc Natl Acad Sci USA; 92:3358-3361.
110.Van Engelen FA, Schouten A, Molthoff JW, et al, 1994. Coordinate expression of antibody subunit genes yields high levels of functional antibodies in roote of transgrnic tobacco, Plant Mol Biol; 26(6): 1701-1710.
111.Van Rooijen GJH & Moloney MM, 1998. Plant seed oil-bodies as carriers for foreign proteins; Biotechnology; 13(1): 72-77.
112.Vander Wilk F, Posthumus D, Husiman MJ, et al, 1991. Expression of the potato leafroll luteovirus cost protein gene in transgenic potato plants inhibits viral infection, Plant Mol Biol; 17(3): 432-439.
113.Visser RG, Jacobsen E, Hasseling-M A, et al, 1989. Transformantion of homozygous diploid potato with an Agrobacterium tumefaciens binary vector system by adventitious shoot regeneration on leaf and stem segments,Plant Mol. Biol.; 12:329-337.
114.Wallis GJ, Dale PJ, Lander D, et al, 1997. Expression of a synthetic antifreeze protein in potato reduces electrolyte release at freezing temperatures. Plant Mol Biol.1997, 35(3):323-30.
115.Wan Y, Lemaux PG, Losonsky G, et al, 1994. Generation of large number of independently transformed fertile barley plants, Plant Physiol.; 104(5):37-48.
116.Wenzler H, Mignery G, May G, et al, 1989. A Rapid and efficient transformation method for the production of large numbers of transgenic potato plants, Plant Sci.; 63: 79-85.
88
117.Whitelam GC, Cockbum B, Verhoeven HA, et al, 1993、Heterologous protein production in transgenic plants, Biotechnology and Genetic Engineering Reviews; 11:1-29.
118.Wilde C, Neven M, Wilde C, et al, 1996. Intact antigen binding MAK33 antibody and fab fragment accumulate in intercellular space of Arabidopsis thaliana, Plant Sci.; 114(2):133-141.
119.Zaenen I Vanlarebeke N, 1974. Supercoiled circular DNA in crown-gall inducing agrobactrium strains, J. Mol Biol.; 86:109-127.
120.Zambryski P & Boston RS, 1992. Chronicles from the Agrobacterium-plant cells DNA transfer story, Ann Rev Plant Physiol; 43: 465-490.
121.Zuo WN, Weissinger AK, Boston RS, et al, 1995. Expression of a maize ribosomeinactivating protein gene in transgenic tobacco plants, Plant Physial Suppl.; 108(2): 150-152.
122.Kodihalli S, Goto H, Darwyn L.K, et al, 1999. DNA Vaccine Encoding Hemagglutinin Provides Protective Immunity against H5N1 Influenza Virus Infection in Mice, Journal of Virology; 73(3):2094-2098.
123．于静娟、敖光明、陈章良等，1997。水稻10KD富硫醇溶蛋白基因在马铃薯中的表达，植物学报；39(4)：329-334。
124．催武、刘炜、吴光耀等，1995。高效、快速地将外源DNA导入根癌土壤杆菌，生物工程学报；11(4)：350—355。
125．刘信、李宁1999。农业生物技术发展与基因工程的安全管理，农业科技管理；99(2)：7～9。
126．刘昕、傅荣昭、蔡民等，1998。SAR与植物转基因沉默的消除，生物工程进展；18(4)：53—58。
127．刘玉乐、田波，1995。 人乙肝病毒表面抗原基因在转基因烟草中的表达，中国科学(B辑)；23：252—255。
128．朱常香、潘春欣，1996。利用PIG基因枪进行水稻转化的研究，山东农业大学学报；27(2)：124—128。
129．朱桢、敖光明、陈章良等，1992。转基因水稻植株再生及外源人α-干扰素cDNA的表达，中国科学(B辑)：22：149-155。
130．王学、王海涛，1996。抗体库的发展及未来，生物化学与生物物理进展；23(4)：312～316。
131．王关林、方宏筠，1998。《植株基因工程原理与技术》，科学出版社；p：46—49。
132．贾士荣、曹冬孙，1992。转基因植物，植物学通报：9(2)：3—15。
133．赵倩、敖光明、于静娟等，1995。牛生长激素基因在马铃薯中的表达，植物学报；37(11)：840-847。
134．雷茂良、程金根，1998。全球转基因植物发展现状，生物技术通报：98 (6)：30～32。
135．刘涤，1999。转基因植物-生产药物的新型反应器，生物技术通报；3：23-27。
136．王新国、肖成祖，1998。用转基因植物生产基因工程疫苗，生物工程进展；18(1)：51-54。
137．顾红雅，翟礼嘉主译，(1998)《植物分子生物学-实验手册》高等教育出版社，北京。
138．金冬雁，黎孟枫等译(1992)《分子克隆》(第二版)，科学出版社，北京。