利用基因工程途径对甘蓝型油菜抗病、抗衰老、无花瓣的研究
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摘要
油菜是我国重要的油料作物，具有十分重要的经济意义。长江中下游地区作为我国油菜的主产区，4～5月油菜生育后期常遇高温多雨天气，菌核病发生严重，经济损失重大。来自于烟草的葡聚糖酶(Glucanase)基因，其编码产物能降解真菌的细胞壁结构成分葡聚糖。分子量为24kDa的植物蛋白AP24则属于细胞程序性死亡(PCD)的诱导蛋白渗透蛋白，对多种真菌也具明显抑制作用。将两基因分别与花椰菜花叶病毒35S启动子相连，根癌农杆菌介导法转化双低甘蓝型油菜品种6-203。对转基因T_2代和T_3代油菜经过田间两种方法的接种鉴定，即苗期离体叶接种和成株期茎杆插签，结合分子鉴定，筛选出外源转基因纯合的株系。
尝试新的转基因方法—活体转化法转化油菜，将自我调控叶片衰老系统P_(SAG12)-ipt融合基因以及gagal基因活体转化法分别转入华双3号，6-203。转基因获得的大量种子首先经卡那霉素(Km)筛选培养基快速筛选，而后继代培养，转入温室，移栽到大田。田间考察形态学性状，结合分子鉴定通过导入P_(SAG12)-ipt以延缓油菜功能叶片的衰老，延长持绿期；通过转入gagal基因旨在获得无花瓣油菜，通过降低油菜菌核病再次传播率达到抗病目的。
这三种转基因油菜的获得，将为抗病育种和抗衰老育种奠定基础。
本研究获得的主要结果如下：
1．油菜菌核病抗性的鉴定方法一定程度决定对油菜抗(耐)菌核病性的判断。通过两种接种方法的多次尝试和比较发现两种接种方法各有优缺点，综合起来作为判断油菜抗(耐)菌核病性的指标才是科学合理的。
2．尝试利用活体转化方法(in planta)转化油菜，证明该法在油菜上是可行的。此法不需经过组织培养，原位进行植物转化。转化效率可以满足基因转移及表达检测的需要。经PCR初步鉴定获得了转基因植株，并表达了预期性状。
3．转基因T_2代理应筛选出纯合株系。转基因抗病材料接种结果显示：在22个株系中发现一个株系有较强的抗病性，接种病斑直径比对照显著减小。初步估计是纯合株系，进一步的反转录PCR(RT-PCR)表明外源基因在植物体内发生转录。该株系T_3代又在两个时期的接种中表现很好，同样进行的RT-PCR和Northern blot都表明外源基因发生体内转录。
4．田间调查转基因抗衰老材料T_0代的单株，着重考察花期后每间隔一周株高和绿叶数的变化趋势，收获和进行考种，考察项目包括：茎粗，一次分枝数，二次分枝数，分枝部位，主枝荚果数，整枝荚果数，每果粒数，千粒重，单株产量，生物学产量等。结果发现，转基因植株生育后期叶片的持绿性显著延长；株高变化范围1.23～1.83m，单株荚果数(反应花总数量)变幅在161～605，千粒重变幅2.27～3.95g生物学产量21.4～
104.229。T:代选到一个优良株系，在Tl代表现良好，叶片持绿期延长，有很强的营养
生长优势，lr[j目PCR鉴定为阳性。T:代时该株系内每一单株无一例外的表现一致，包
括生育期，I]一十片持绿时间等，证明为一个优良株系。
5.转基因无花瓣油菜由于环境(光照，气温)等因素综合影响，表型很难稳定遗传。
仅在T。代观察到无花瓣现象，但这些无花瓣材料往往发生许多不利性状的突变，诸如
植株矮化，株型矮缩，花序无分枝，育性减弱:初花期雌蕊外露，盛花期花量极少;不
能正常结果，套袋自交后少数能结实，但结实性低，多数是瘪粒。这些突变产生的原因
可能是T一DNA插入植物基因组时，引起植物基因组的重排，导致不良睦状的产生。此外，
外源基因插入的位点及插入拷贝数复杂性也影响其表达。
Sclerotinia stem rot that caused by Sclerotinia sclerotiorum is the most devastating disease of rapeseed (Brassica napus L.) in China especially in Yangtze River Area and leads to great yield loss every year. Glucanase and AP24, which come from tobacco both, have broad spectrum of antifungal activity. AP24 and Glu were combined with CaMV35S promoter and were transferred into Brassica napus via Agrobacterium tumefaciens. We took two different inoculation methods during two different periods to estimate the resistance to Sclerotinia sclerotiorum of T2 and Ta progenies. That is the detached leaves inoculation and inoculation of stem by tooth sticks clinged with pathogene. The plants exhibited an increased resistance against the fungal attack compared with the no transgenic plants during two inoculations. The candidate plants were conducted with RT-PCR and Northern blot analysis. The result indicated the increase in the transcriptional level of foreign genes.
A method for in situ transformation-in planta transformation was also preceded. We attempted this new transformation method on a variety of Brassica napus, Hua Shuang No.3, and collected 1,800 seed grains after treated with Agrobacterium tumefaciens containing the chimeric PsAGn-IPT gene. The harveated seeds were screened on MS medium with Kanamycine. The Km -resistant seedlings were transplanted into soil in a greenhouse then in the field and developed into 70 adult plants. So, 3.8% of in-planta seeds were grown to mature plants. PCR analysis showed that 16 among 70 plants had been integrated into the PsAGn-IPT. Several agronomy traits on the 16 TO plants were investigated including the plant height, green leaf number on main stems, flower number, biomass, seed yield and weight of seed grains etc. The highest green leaf number that the plant can reached of each plant varied much, from 15 to 56 among the 16 plants. The plant height ranged from 1.23 to 1.83m. The weight of seed grains varied from 2.27 to 3.95g/1000grains. The flower number ranged from 161 to 605. Biomass changed much from 21.4 to 104.22g. We chosed a nicer plant from TO generation, and the following field investigation also displayed favorable characters such as the green leaf number, biomass etc.
With the same in planta transformation method we got the transgenic gagal plants. During the flowering time the apetalous flower can be found. But some bad characters were detected at the same time, which was depicted as following: the shorter plant shape, the fewer branch, little flower, less seed yield et al.
尝试新的转基因方法—活体转化法转化油菜，将自我调控叶片衰老系统P_(SAG12)-ipt融合基因以及gagal基因活体转化法分别转入华双3号，6-203。转基因获得的大量种子首先经卡那霉素(Km)筛选培养基快速筛选，而后继代培养，转入温室，移栽到大田。田间考察形态学性状，结合分子鉴定通过导入P_(SAG12)-ipt以延缓油菜功能叶片的衰老，延长持绿期；通过转入gagal基因旨在获得无花瓣油菜，通过降低油菜菌核病再次传播率达到抗病目的。
这三种转基因油菜的获得，将为抗病育种和抗衰老育种奠定基础。
本研究获得的主要结果如下：
1．油菜菌核病抗性的鉴定方法一定程度决定对油菜抗(耐)菌核病性的判断。通过两种接种方法的多次尝试和比较发现两种接种方法各有优缺点，综合起来作为判断油菜抗(耐)菌核病性的指标才是科学合理的。
2．尝试利用活体转化方法(in planta)转化油菜，证明该法在油菜上是可行的。此法不需经过组织培养，原位进行植物转化。转化效率可以满足基因转移及表达检测的需要。经PCR初步鉴定获得了转基因植株，并表达了预期性状。
3．转基因T_2代理应筛选出纯合株系。转基因抗病材料接种结果显示：在22个株系中发现一个株系有较强的抗病性，接种病斑直径比对照显著减小。初步估计是纯合株系，进一步的反转录PCR(RT-PCR)表明外源基因在植物体内发生转录。该株系T_3代又在两个时期的接种中表现很好，同样进行的RT-PCR和Northern blot都表明外源基因发生体内转录。
4．田间调查转基因抗衰老材料T_0代的单株，着重考察花期后每间隔一周株高和绿叶数的变化趋势，收获和进行考种，考察项目包括：茎粗，一次分枝数，二次分枝数，分枝部位，主枝荚果数，整枝荚果数，每果粒数，千粒重，单株产量，生物学产量等。结果发现，转基因植株生育后期叶片的持绿性显著延长；株高变化范围1.23～1.83m，单株荚果数(反应花总数量)变幅在161～605，千粒重变幅2.27～3.95g生物学产量21.4～
104.229。T:代选到一个优良株系，在Tl代表现良好，叶片持绿期延长，有很强的营养
生长优势，lr[j目PCR鉴定为阳性。T:代时该株系内每一单株无一例外的表现一致，包
括生育期，I]一十片持绿时间等，证明为一个优良株系。
5.转基因无花瓣油菜由于环境(光照，气温)等因素综合影响，表型很难稳定遗传。
仅在T。代观察到无花瓣现象，但这些无花瓣材料往往发生许多不利性状的突变，诸如
植株矮化，株型矮缩，花序无分枝，育性减弱:初花期雌蕊外露，盛花期花量极少;不
能正常结果，套袋自交后少数能结实，但结实性低，多数是瘪粒。这些突变产生的原因
可能是T一DNA插入植物基因组时，引起植物基因组的重排，导致不良睦状的产生。此外，
外源基因插入的位点及插入拷贝数复杂性也影响其表达。
Sclerotinia stem rot that caused by Sclerotinia sclerotiorum is the most devastating disease of rapeseed (Brassica napus L.) in China especially in Yangtze River Area and leads to great yield loss every year. Glucanase and AP24, which come from tobacco both, have broad spectrum of antifungal activity. AP24 and Glu were combined with CaMV35S promoter and were transferred into Brassica napus via Agrobacterium tumefaciens. We took two different inoculation methods during two different periods to estimate the resistance to Sclerotinia sclerotiorum of T2 and Ta progenies. That is the detached leaves inoculation and inoculation of stem by tooth sticks clinged with pathogene. The plants exhibited an increased resistance against the fungal attack compared with the no transgenic plants during two inoculations. The candidate plants were conducted with RT-PCR and Northern blot analysis. The result indicated the increase in the transcriptional level of foreign genes.
A method for in situ transformation-in planta transformation was also preceded. We attempted this new transformation method on a variety of Brassica napus, Hua Shuang No.3, and collected 1,800 seed grains after treated with Agrobacterium tumefaciens containing the chimeric PsAGn-IPT gene. The harveated seeds were screened on MS medium with Kanamycine. The Km -resistant seedlings were transplanted into soil in a greenhouse then in the field and developed into 70 adult plants. So, 3.8% of in-planta seeds were grown to mature plants. PCR analysis showed that 16 among 70 plants had been integrated into the PsAGn-IPT. Several agronomy traits on the 16 TO plants were investigated including the plant height, green leaf number on main stems, flower number, biomass, seed yield and weight of seed grains etc. The highest green leaf number that the plant can reached of each plant varied much, from 15 to 56 among the 16 plants. The plant height ranged from 1.23 to 1.83m. The weight of seed grains varied from 2.27 to 3.95g/1000grains. The flower number ranged from 161 to 605. Biomass changed much from 21.4 to 104.22g. We chosed a nicer plant from TO generation, and the following field investigation also displayed favorable characters such as the green leaf number, biomass etc.
With the same in planta transformation method we got the transgenic gagal plants. During the flowering time the apetalous flower can be found. But some bad characters were detected at the same time, which was depicted as following: the shorter plant shape, the fewer branch, little flower, less seed yield et al.
引文
1．于晓红，朱勇清，许智宏等。种子特异表达ipt转基因棉花根和纤维的改变。植物学报，2000，42(1)：59～63
2．马庆虎等。异戊烯基转移酶基因在转基因烟草中的特异性表达 植物学报，1994，36(5)：339-344
3．孔振辉，Milner JJ．拟南芥基因转移新方法-真空渗入法的研究，西北植物学报，1996，16(3)：277-283
4．王三根。细胞分裂素在植物抗逆和延缓衰老中的作用，植物学通报，2000，17(2)：121-126
5．王关林，方宏筠主编。植物基因工程原理和技术。科学出版社，1998
6．王槐，陈正华。基质结合区(MARs)与转基因植物的基因表达。生命科学 1999 11(2)：54-57
7．冯道荣，李宝健等。使用双抗真菌蛋白基因提高水稻抗病性的研究。植物学报，1999，41(11)
8．叶健明，唐克轩等。植物转基因方法概述，生命科学，1999，11(2)：58-60
9．左开井。转基因抗虫棉Bt基因的遗传效应及定位研究，华中农业大学博士论文 2000。
10．田桂英。向植物导入外源DNA方法的研究与发展 生物工程进展，1996，2：4-7
11．刘后利著。油菜遗传育种学 中国农业大学出版社 2001
12．刘丽君，吴俊江，高明杰等。转ipt基因大豆感染SMV1号的生理特性变化。大豆科学，1999，18(3)：213～217
13．刘道宏。植物叶片的衰老，植物生理学通讯，1983(2)：14-19
14．华志华，黄大年。转基因植物中外源基因的遗传学行为，植物学报，1999，41(1)1-5
15．华志华，黄大年。植物转基因沉默的研究与对策。生命科学 1999 11(2)：51-53
16．华志华，黄大年等。基因枪转化获得的转基因水稻中外源基因整合与表达规律研究，遗传学报 28(11)：2001，1012-1018
17．朱祯，李向辉。植物遗传工程研究进展 生物工程进展，1991，11(5)：35-43
18．朱莉，张春义，范云六。转基因植物中外源基因沉默机制的研究进展。生物化学与生物物理进展，1999 1：
19．许智宏。植物发育与生殖的研究：进展和展望，植物学报 1999，41(9)
20．许耀等。1991，实验生物学报 24(2)：109-115
21．吴刚，夏英武。植物转基因沉默及对策。生物技术，2000，10(2)：27-32
22．张钰，Adrian J，Calter．真空渗透法转化植物的研究。生物技术，1999 9(2)：35
-36
23．张赛群，叶志彪等。番茄异戊烯基转移酶的遗传转化及转基因植株再生。园艺学报，1999，26(6)：376～379
24．张赛群。番茄异戊烯基转移酶的遗传转化及转基因植株再生 华中农业大学硕士毕业论文，1998
25．李子银，胡会庆。农杆菌介导的植物遗传转化进展 1998生物工程进展 18(1)：22-26
26．李文风，吴立人等。植物抗病基因克隆与功能研究进展。生命科学 2001，13(4)：151-153
27．李汝刚 范云六。植物抗菌物病害的遗传工程研究进展 生物工程进展 2000，20(2)：9-13
28．李宏，王新力。植物组织RNA提取的难点及对策。生物技术通报，1999，1：36-39
29．李学宝，秦明辉等。芥菜型油菜抗虫转基因植株及其后代株系的研究，生物工程学报，1999 15(4)：482-488
30．杨金水等。1991植物学报，33(1)：825-832
31．杨剑波等。1993实验生物学报 26(1)：1-7
32．沈成国，张福锁，毛达如。植物叶片衰老过程中叶绿素降解代谢研究进展 植物学通报 1998，15(增刊)：41-46
33．陈正华等。抗菌核病转基因油菜的获得 云南大学学报(自然科学版)2000：177-178
34．陈玉卿，张洁夫，吕忠进。甘蓝型无花瓣油菜对菌核病抗(耐)性研究，江苏农业科学 1991，6：10-13
35．洪剑明，印莉萍，邱泽生。植物抗病基因工程进展(续)植物学通报 1998，15(1)：8-16
36．赵建伟 孟金陵。植物抗病基因的克隆及其在作物遗传改良中的意义 高技术通讯 1998：8(5)：58-62
37．夏兰芹，郭三堆等。2000，外源基因在转基因植物中的表达与稳定性，生物技术通报，3：8-12
38．耿，麻密等。ipt基因定位表达对转基因烟草育性的影响。植物学报，2000，42(2)：217～220
39．曹孟良。农杆菌介导的水稻遗传转化及自我调控延缓叶片衰老系统在水稻中的表现。华中农业大学博士毕业论文，1998
40．黄和平，叶志彪。农业高新技术产业化研究，武汉出版社，2000
41．傅寿仲等。油菜无花瓣育种的研究进展 中国油料 1992，1：1-6
42．蓝海燕，陈正华。植物与病原真菌互作的分子生物学及其研究进展 生物工程进展 2000 20(4)：16-22
43．蓝海燕，陈正华。农杆菌介导的芸薹属植物遗传转化技术的研究进展 1999，生物技
术通报，3：15-18
44．蓝海燕，陈正华。葡聚糖酶及其在植物中的发育调节和防卫反应。生物技术通报 1998，4：10-15
45．蓝海燕，陈正华等。农杆菌介导法将β-1，3葡聚糖酶基因导入油菜的研究初报 中国油料作物学报 2000 22(1)：6-10
46．精编分子生物学实验指南，[美]F．奥斯伯等著，王海林译，科学出版社，1998
47．谭小力，李殿荣等。甘蓝型油菜无花瓣品系的选育研究，西北农业大学学报。1998，26(3)27-31
48．孙万仓。转基因油菜研究进展 西北农业学报 2000，9(3)：117-121
49．陈锦清，郎春秀。反义PEP基因调控油菜籽粒蛋白质／油脂含量比率的研究 农业生物技术学报 1999，7(4)：316-320
50．郎春秀，胡张华。油菜农杆菌转基因体系的建立及转PEP反义基因油菜的获得 浙江农业学报 1999，11(2)：55-58
51．候丙凯，陈正华等。激光微束穿刺法获得抗虫转基因油菜及其后代的研究 中国激光 2001，28(1)：93-96
52. A model for RNA-mediated gene silencing in higher plants Plant molecular Biology
53. Ainsworth C. Isolation of RNA from floral tissue of Rumex acetosa(sorrel). Plant Mol Biol Reporter, 1994, 12: 198-203
54. Albert H, Dale E C, Lee E, Ow D W. Site-specific integration of DNA into wild-type and mutant Lox sites placed in the plant genome. Plant J, 1995, 7: 649～659
55. Broglie R, Broglie K: Production of disease-resistance transgenic plants. Current Opinion in Biotechnology 1993, 4: 148-151.
56. Budar F, Thia-Toong L,Van Montagr M, Hernalsteens J P. Agrobacterium-mediated gene transfer results mainly in transgenic plants transmitting T-DNA as a single Mendelian factor. Genetics, 1986, 114: 303～313
57. Buzza GC. The inheritance of an apetalous flower character in Canola. Cruciferae Newsletter 1983, 8: 11-12
58. Christou P, Swain W F, Yang N S, McCabe D E. Inheritance and expression of foreign genes in transgenic soybean plants. Proc Natl Acad Sci USA, 1989, 86: 7500～7504
59. Christou P. Strategies for variety-independent genetic transformation of important cereals, legumes and woody species utilizing particle bombardment. Euphytica, 1995, 85: 13～27
60. Coen E S, Carpenter R.. The metamorphosis of flowers Plant Cell, 1998, 5: 1115-1181
61. Coen E S, Meyerowitz E M, The war of the whorls: Genetic interactions controlling flower development, Nature, 1991, 63: 1311-1322
62. De carvalho Niebel F et al, EMBD J, 1992 11: 2595-5602
63. Deroles S C, Gardner R C. Expression and inheritance of kanamycin resistance in a large number of transgenic petunias generated by Agrobacterium-mediated transformation. Plant Mol Biol, 1988, 11: 355～364
64. Elmayan T, Vaucheret H. Plant J, 1996, 9: 787-797
65. Fary MJ, Erans E. J. et al, Physiological assessment of apetalous flowers and erectophile pods in oilseed rape. Journal of Agricultural Science 1996, 127: 193-200
66. Finnegan J, McElory D. Transgene inactivation: plants fight back! Bio. Technology, 1994, 12: 883-888.
67. Gan Shusheng, Amsaino R. Cytokinin in plant senescence from spay and pray to clone and play. Bioessay, 1996, 18: 557～565
68. Gan Shusheng, Amsaino R. Inhibition of leaf senescence by autoregulated production of cytokinin, Science, 1995, 270: 1986～1988
69. Gan Shusheng, Amsaino R. Making sense of senescence molecular regulation of leaf senescence. Plant Physiology, 1997, 113: 313～319
70. Grison R et al., Field tolerance to fungal pathogens of Brassica napus constitutively expressing a chimeric chitinase gene Nature Biotechnology, 1996, 14: 643-646
71. Guolan Wei, Deyue yu et al., Transformation of soybean with floral regulatory gene gagal from Gerbera Hybrida Plant Genomics in China □ 2001: 123
72. Hart CM., Fischer B Meins Jr F. Mol Gen Genet, 1992, 235: 179-188
73. Herrera-Esfrella L, Van den Broeck G, Maenaut R, Van Montagu M, Schell J, Timko M, Cashmore A. Light-inducible and chloroplast-associated expression of a chimeric gene into Nicotiana tabacum using a Ti plasmid vector. Nature, 1984, 310: 115～120
74. Herrera-Esfrella L, Van den Broeck G, Maenaut R, Van Montagu M, Schell J, Timko M, Cashmore A. Light-inducible and chloroplast-associated expression of a chimeric gene into Nicotiana tabacum using a Ti plasmid vector. Nature, 1984, 310: 115～120
75. Iyer LM et al, Transgene silencing in monocots. Plant Mol Biol, 2000, 43: 323-346
76. Ismail Serageldin Biotechnology and Food Security in the 21st Century. Science, 1999, 285: 387-389
77. Kumpatla S P, Chandrasekharan MB, Lyer L M, et al. Genome inruder scanning and modulation systems and transgene silencingl Trends in Plant Science, 1998, 3(3): 97-104
78. Lebel E G, Masson J, Bogucki A, Paszkowski J. Transposable elements as plant transformation vectors for long stretches of foreign DNA. Thero Appl Genet, 1995, 91: 899～906
79. Napoli C, Lemieux C, Jorgensen R. Introduction of a chimeric chaloone synthase gene into
perunia results in reversile co-suppression of homologous genes in trans. Plant Cell, 1990, 23: 279～289
80. Paolo Amedeo et al., Disruption of the plant gene MOM releases transcriptional silencing of methylated genes 2000 Nature405: 203-206
81. Peerbolte R, Leenhouts K, Hooykaas-van Slogteren G M S, et al. Clone from a shooty tobacco crown gall tumor irregular T-DNA structures and organization T-DNA, methylation and conditional expression of opine genes. Plant Mol Bio. 1986, 7: 285-299
82. Powell A P et al Delay of disease developmant in transgenic plants that express the tobacco mosaic virus coat protein gene, Science 1986 232: 738-743
83. Prinsen E, et al, J. Plant Physiol 1994, 144: 80-85
84. Sanford J. C, Klein TM, et al, Particulate Science and Technology, 1987, (5): 27-37
85. Schocher R J, Shillito R D, Saul M W, Paszkowski, Potrykus I. Co-transformation of unlinked foreign genes into plants by direct gene transfer. Bio/Technology, 1986, 4: 1093～1096
86. Shimamoto K, Terada R, Izawa T, Fujimoto H. Fertile transgenic rice plants regenerated from transformed protoplasts. Nature, 1989, 338: 274～276
87. Shimamoto K, Terada R, Izawa T, Fujimoto H. Fertile transgenic rice plants regenerated from transformed protoplasts. Nature, 1989, 338: 274～276
88. Soraya Pelaz et al., B and C floral organ identity functions require Soraya SEPALLATA MADS-box genes 2000 Nature 405: 200-203
89. Spencer T M, O'Brien J V, Start W G, Adams T R, Gordon-Kamm W J, Lemaux P G. Segregation of transgenes in maize. Plant Mol Biol, 1992, 18: 201～210
90. Spiker S, Thompson W F. Nuclear matrix attachment regions and transgene expression in plants. Plant Physiol, 1996, 110: 15～21
91. Susheng Gan, Richard M. Amasino. Inhibition of leaf senescence by autoregulated production of cyto-kinin Science 1995, 270: 1986-1988
92. Toriyama K, Arimoto Y, Uchimiya H, Hinata K. Transgenic rice plants after direct gene transfer into protoplasts. Bio/Technology, 1988, 6: 1072～1074
93. Toriyama K, Arimoto Y, Uchimiya H, Hinata K. Transgenic rice plants after direct gene transfer into protoplasts. Bio/Technology, 1988, 6: 1072～1074
94. Wright SC, et al.,: Tumor cell resistance to apoptosis due to a defect in the activation of sphingomyelinase and the 24 KDa apoptotic protease(AP24). FASEB J 1996 Feb; 10(2): 325-329
95. Wubben JP, et al. Physiol Mol Pathol, 1992, 41(1): 23-32
96. Zambryski P. Basic processes underlying Agrobacterium-mediated DNA transfer to plant
cells. Annu Rev Genet, 1988, 22: 1～30
97. Zhu Qun et al., Enhanced protection against fungal attack by constitutive co-expression of chitinase and glucanase genes in transgenic tobacco. Biotechnology 1994, 12: 807-812
2．马庆虎等。异戊烯基转移酶基因在转基因烟草中的特异性表达 植物学报，1994，36(5)：339-344
3．孔振辉，Milner JJ．拟南芥基因转移新方法-真空渗入法的研究，西北植物学报，1996，16(3)：277-283
4．王三根。细胞分裂素在植物抗逆和延缓衰老中的作用，植物学通报，2000，17(2)：121-126
5．王关林，方宏筠主编。植物基因工程原理和技术。科学出版社，1998
6．王槐，陈正华。基质结合区(MARs)与转基因植物的基因表达。生命科学 1999 11(2)：54-57
7．冯道荣，李宝健等。使用双抗真菌蛋白基因提高水稻抗病性的研究。植物学报，1999，41(11)
8．叶健明，唐克轩等。植物转基因方法概述，生命科学，1999，11(2)：58-60
9．左开井。转基因抗虫棉Bt基因的遗传效应及定位研究，华中农业大学博士论文 2000。
10．田桂英。向植物导入外源DNA方法的研究与发展 生物工程进展，1996，2：4-7
11．刘后利著。油菜遗传育种学 中国农业大学出版社 2001
12．刘丽君，吴俊江，高明杰等。转ipt基因大豆感染SMV1号的生理特性变化。大豆科学，1999，18(3)：213～217
13．刘道宏。植物叶片的衰老，植物生理学通讯，1983(2)：14-19
14．华志华，黄大年。转基因植物中外源基因的遗传学行为，植物学报，1999，41(1)1-5
15．华志华，黄大年。植物转基因沉默的研究与对策。生命科学 1999 11(2)：51-53
16．华志华，黄大年等。基因枪转化获得的转基因水稻中外源基因整合与表达规律研究，遗传学报 28(11)：2001，1012-1018
17．朱祯，李向辉。植物遗传工程研究进展 生物工程进展，1991，11(5)：35-43
18．朱莉，张春义，范云六。转基因植物中外源基因沉默机制的研究进展。生物化学与生物物理进展，1999 1：
19．许智宏。植物发育与生殖的研究：进展和展望，植物学报 1999，41(9)
20．许耀等。1991，实验生物学报 24(2)：109-115
21．吴刚，夏英武。植物转基因沉默及对策。生物技术，2000，10(2)：27-32
22．张钰，Adrian J，Calter．真空渗透法转化植物的研究。生物技术，1999 9(2)：35
-36
23．张赛群，叶志彪等。番茄异戊烯基转移酶的遗传转化及转基因植株再生。园艺学报，1999，26(6)：376～379
24．张赛群。番茄异戊烯基转移酶的遗传转化及转基因植株再生 华中农业大学硕士毕业论文，1998
25．李子银，胡会庆。农杆菌介导的植物遗传转化进展 1998生物工程进展 18(1)：22-26
26．李文风，吴立人等。植物抗病基因克隆与功能研究进展。生命科学 2001，13(4)：151-153
27．李汝刚 范云六。植物抗菌物病害的遗传工程研究进展 生物工程进展 2000，20(2)：9-13
28．李宏，王新力。植物组织RNA提取的难点及对策。生物技术通报，1999，1：36-39
29．李学宝，秦明辉等。芥菜型油菜抗虫转基因植株及其后代株系的研究，生物工程学报，1999 15(4)：482-488
30．杨金水等。1991植物学报，33(1)：825-832
31．杨剑波等。1993实验生物学报 26(1)：1-7
32．沈成国，张福锁，毛达如。植物叶片衰老过程中叶绿素降解代谢研究进展 植物学通报 1998，15(增刊)：41-46
33．陈正华等。抗菌核病转基因油菜的获得 云南大学学报(自然科学版)2000：177-178
34．陈玉卿，张洁夫，吕忠进。甘蓝型无花瓣油菜对菌核病抗(耐)性研究，江苏农业科学 1991，6：10-13
35．洪剑明，印莉萍，邱泽生。植物抗病基因工程进展(续)植物学通报 1998，15(1)：8-16
36．赵建伟 孟金陵。植物抗病基因的克隆及其在作物遗传改良中的意义 高技术通讯 1998：8(5)：58-62
37．夏兰芹，郭三堆等。2000，外源基因在转基因植物中的表达与稳定性，生物技术通报，3：8-12
38．耿，麻密等。ipt基因定位表达对转基因烟草育性的影响。植物学报，2000，42(2)：217～220
39．曹孟良。农杆菌介导的水稻遗传转化及自我调控延缓叶片衰老系统在水稻中的表现。华中农业大学博士毕业论文，1998
40．黄和平，叶志彪。农业高新技术产业化研究，武汉出版社，2000
41．傅寿仲等。油菜无花瓣育种的研究进展 中国油料 1992，1：1-6
42．蓝海燕，陈正华。植物与病原真菌互作的分子生物学及其研究进展 生物工程进展 2000 20(4)：16-22
43．蓝海燕，陈正华。农杆菌介导的芸薹属植物遗传转化技术的研究进展 1999，生物技
术通报，3：15-18
44．蓝海燕，陈正华。葡聚糖酶及其在植物中的发育调节和防卫反应。生物技术通报 1998，4：10-15
45．蓝海燕，陈正华等。农杆菌介导法将β-1，3葡聚糖酶基因导入油菜的研究初报 中国油料作物学报 2000 22(1)：6-10
46．精编分子生物学实验指南，[美]F．奥斯伯等著，王海林译，科学出版社，1998
47．谭小力，李殿荣等。甘蓝型油菜无花瓣品系的选育研究，西北农业大学学报。1998，26(3)27-31
48．孙万仓。转基因油菜研究进展 西北农业学报 2000，9(3)：117-121
49．陈锦清，郎春秀。反义PEP基因调控油菜籽粒蛋白质／油脂含量比率的研究 农业生物技术学报 1999，7(4)：316-320
50．郎春秀，胡张华。油菜农杆菌转基因体系的建立及转PEP反义基因油菜的获得 浙江农业学报 1999，11(2)：55-58
51．候丙凯，陈正华等。激光微束穿刺法获得抗虫转基因油菜及其后代的研究 中国激光 2001，28(1)：93-96
52. A model for RNA-mediated gene silencing in higher plants Plant molecular Biology
53. Ainsworth C. Isolation of RNA from floral tissue of Rumex acetosa(sorrel). Plant Mol Biol Reporter, 1994, 12: 198-203
54. Albert H, Dale E C, Lee E, Ow D W. Site-specific integration of DNA into wild-type and mutant Lox sites placed in the plant genome. Plant J, 1995, 7: 649～659
55. Broglie R, Broglie K: Production of disease-resistance transgenic plants. Current Opinion in Biotechnology 1993, 4: 148-151.
56. Budar F, Thia-Toong L,Van Montagr M, Hernalsteens J P. Agrobacterium-mediated gene transfer results mainly in transgenic plants transmitting T-DNA as a single Mendelian factor. Genetics, 1986, 114: 303～313
57. Buzza GC. The inheritance of an apetalous flower character in Canola. Cruciferae Newsletter 1983, 8: 11-12
58. Christou P, Swain W F, Yang N S, McCabe D E. Inheritance and expression of foreign genes in transgenic soybean plants. Proc Natl Acad Sci USA, 1989, 86: 7500～7504
59. Christou P. Strategies for variety-independent genetic transformation of important cereals, legumes and woody species utilizing particle bombardment. Euphytica, 1995, 85: 13～27
60. Coen E S, Carpenter R.. The metamorphosis of flowers Plant Cell, 1998, 5: 1115-1181
61. Coen E S, Meyerowitz E M, The war of the whorls: Genetic interactions controlling flower development, Nature, 1991, 63: 1311-1322
62. De carvalho Niebel F et al, EMBD J, 1992 11: 2595-5602
63. Deroles S C, Gardner R C. Expression and inheritance of kanamycin resistance in a large number of transgenic petunias generated by Agrobacterium-mediated transformation. Plant Mol Biol, 1988, 11: 355～364
64. Elmayan T, Vaucheret H. Plant J, 1996, 9: 787-797
65. Fary MJ, Erans E. J. et al, Physiological assessment of apetalous flowers and erectophile pods in oilseed rape. Journal of Agricultural Science 1996, 127: 193-200
66. Finnegan J, McElory D. Transgene inactivation: plants fight back! Bio. Technology, 1994, 12: 883-888.
67. Gan Shusheng, Amsaino R. Cytokinin in plant senescence from spay and pray to clone and play. Bioessay, 1996, 18: 557～565
68. Gan Shusheng, Amsaino R. Inhibition of leaf senescence by autoregulated production of cytokinin, Science, 1995, 270: 1986～1988
69. Gan Shusheng, Amsaino R. Making sense of senescence molecular regulation of leaf senescence. Plant Physiology, 1997, 113: 313～319
70. Grison R et al., Field tolerance to fungal pathogens of Brassica napus constitutively expressing a chimeric chitinase gene Nature Biotechnology, 1996, 14: 643-646
71. Guolan Wei, Deyue yu et al., Transformation of soybean with floral regulatory gene gagal from Gerbera Hybrida Plant Genomics in China □ 2001: 123
72. Hart CM., Fischer B Meins Jr F. Mol Gen Genet, 1992, 235: 179-188
73. Herrera-Esfrella L, Van den Broeck G, Maenaut R, Van Montagu M, Schell J, Timko M, Cashmore A. Light-inducible and chloroplast-associated expression of a chimeric gene into Nicotiana tabacum using a Ti plasmid vector. Nature, 1984, 310: 115～120
74. Herrera-Esfrella L, Van den Broeck G, Maenaut R, Van Montagu M, Schell J, Timko M, Cashmore A. Light-inducible and chloroplast-associated expression of a chimeric gene into Nicotiana tabacum using a Ti plasmid vector. Nature, 1984, 310: 115～120
75. Iyer LM et al, Transgene silencing in monocots. Plant Mol Biol, 2000, 43: 323-346
76. Ismail Serageldin Biotechnology and Food Security in the 21st Century. Science, 1999, 285: 387-389
77. Kumpatla S P, Chandrasekharan MB, Lyer L M, et al. Genome inruder scanning and modulation systems and transgene silencingl Trends in Plant Science, 1998, 3(3): 97-104
78. Lebel E G, Masson J, Bogucki A, Paszkowski J. Transposable elements as plant transformation vectors for long stretches of foreign DNA. Thero Appl Genet, 1995, 91: 899～906
79. Napoli C, Lemieux C, Jorgensen R. Introduction of a chimeric chaloone synthase gene into
perunia results in reversile co-suppression of homologous genes in trans. Plant Cell, 1990, 23: 279～289
80. Paolo Amedeo et al., Disruption of the plant gene MOM releases transcriptional silencing of methylated genes 2000 Nature405: 203-206
81. Peerbolte R, Leenhouts K, Hooykaas-van Slogteren G M S, et al. Clone from a shooty tobacco crown gall tumor irregular T-DNA structures and organization T-DNA, methylation and conditional expression of opine genes. Plant Mol Bio. 1986, 7: 285-299
82. Powell A P et al Delay of disease developmant in transgenic plants that express the tobacco mosaic virus coat protein gene, Science 1986 232: 738-743
83. Prinsen E, et al, J. Plant Physiol 1994, 144: 80-85
84. Sanford J. C, Klein TM, et al, Particulate Science and Technology, 1987, (5): 27-37
85. Schocher R J, Shillito R D, Saul M W, Paszkowski, Potrykus I. Co-transformation of unlinked foreign genes into plants by direct gene transfer. Bio/Technology, 1986, 4: 1093～1096
86. Shimamoto K, Terada R, Izawa T, Fujimoto H. Fertile transgenic rice plants regenerated from transformed protoplasts. Nature, 1989, 338: 274～276
87. Shimamoto K, Terada R, Izawa T, Fujimoto H. Fertile transgenic rice plants regenerated from transformed protoplasts. Nature, 1989, 338: 274～276
88. Soraya Pelaz et al., B and C floral organ identity functions require Soraya SEPALLATA MADS-box genes 2000 Nature 405: 200-203
89. Spencer T M, O'Brien J V, Start W G, Adams T R, Gordon-Kamm W J, Lemaux P G. Segregation of transgenes in maize. Plant Mol Biol, 1992, 18: 201～210
90. Spiker S, Thompson W F. Nuclear matrix attachment regions and transgene expression in plants. Plant Physiol, 1996, 110: 15～21
91. Susheng Gan, Richard M. Amasino. Inhibition of leaf senescence by autoregulated production of cyto-kinin Science 1995, 270: 1986-1988
92. Toriyama K, Arimoto Y, Uchimiya H, Hinata K. Transgenic rice plants after direct gene transfer into protoplasts. Bio/Technology, 1988, 6: 1072～1074
93. Toriyama K, Arimoto Y, Uchimiya H, Hinata K. Transgenic rice plants after direct gene transfer into protoplasts. Bio/Technology, 1988, 6: 1072～1074
94. Wright SC, et al.,: Tumor cell resistance to apoptosis due to a defect in the activation of sphingomyelinase and the 24 KDa apoptotic protease(AP24). FASEB J 1996 Feb; 10(2): 325-329
95. Wubben JP, et al. Physiol Mol Pathol, 1992, 41(1): 23-32
96. Zambryski P. Basic processes underlying Agrobacterium-mediated DNA transfer to plant
cells. Annu Rev Genet, 1988, 22: 1～30
97. Zhu Qun et al., Enhanced protection against fungal attack by constitutive co-expression of chitinase and glucanase genes in transgenic tobacco. Biotechnology 1994, 12: 807-812