水稻农林8号、农林8号m cDNA文库的构建和BSL基因的筛选
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摘要
水稻农林8号m是农林8号通过~(60)CO γ射线辐射诱变获得的一个突变体。遗传分析表明：该突变性状表现为隐性单基因的遗传，在苗期能被0.05％以上的苯达松全部杀死，而其它常规品种则表现为对苯达松的抗性。而且，苯达松敏感致死基因对水稻的农艺性状和杂交优势没有不良的影响。将苯达松敏感致死基因转化到不育系或恢复系中，可以用于保证和提高水稻杂种纯度，革新水稻的制种方法。
将约5万粒农林8号m干种子，平均分成12等分，分别用γ射线(0，200，225，250，275，300Gy六个剂量)和离子注入(0，2000，2300，2600，2900，3200六个单位剂量，每个单位剂量约2.6×10~(13)N~+／cm~2)进行诱变处理，处理后的农林8号m发苗至苗二叶期，用0.05％苯达松水培筛选抗性苗，未能获得使农林8号m突变基因功能恢复抗性苗，推测该突变可能不是点突变导致的。
农林8号和农林8号m种子萌发15天左右至苗二叶期。以稻苗为材料，抽提总RNAs，分离mRNAs。分别构建农林8号和农林8号m cDNA文库。初级文库的库容量分别为1.2×10~6，1.4×10~6。随机挑取28个噬菌斑，用T_3、T_7作引物，通过PCR检测，获得插入片断大小从400bp～3kb，平均约1kb。通过x-gal／IPTG蓝白斑鉴定，连接效率99％以上。上述所有皆符合cDNA文库建的总体要求。
参照农林8号和农林8号m RAPD分子标记的结果，设计特异引物，通过PCR获得制备探针的DNA模板。用地高辛标记模板制作探针与农林8号cDNA文库中约30万个克隆子进行Southern原位杂交。获得了一些有杂交信号的噬菌斑，并对目的片断进行克隆和序列分析。
A chemical lethal rice mutant Norin 8 m earring the bentazone sensitive lethal gene was induced from Norin 8 by 60Co gamma-ray radiation. The results indicated that bentazone sensitive lethal gene was controlled by a single recessive gene . The Norin 8 m seedling can be killed by bentazone (above 0.05%), but its FI seedlings are safe. Furthermore, the gene has no negative effects on agronomic traits no matter whether it is homozygous or heterozygous. The techniques for transferring and selecting the marker gene in breeding program and its application ensure the purity of hybrid seed. A making system of the two-line hybrid rice had been put forward.
About 50,000 Norin 8m seeds were divided into 12 parts. The Norin 8m were treated with 0, 200, 225, 250, 275, 300 Gy CO60 and 0, 2000, 2300, 2600, 2900, 3200 X 2. 6X 1013N+/cm2 law energy ion bean implantation respectively. At the 2-leaf stage, all plants were cultived with 0.05% bentazon, the function of mutant gene can't be recovered.
Total RNAs were extracted from seedling of Norin8 and its 60CO gamma-ray irradiation mutant Norin8 m during 2-3 leaves. The messenger RNA was isolated from the total RNA, the Norin 8 and Norin 8 m rice cDNA libraries were constructed. The primary libraies had a total of 1.2 106 phages for Norin 8 and a total 1.4 106 phages for Norin 8 m, 28 randomly selected clones were evaluated. Insert fragments by PCR amplification using T3, T7 primer were obtained and ranged from 400bp to 3kb with an average of 1kb. The 99% phages were recombinants by x-gal/IPTG. All of the above mentioned accorded with the general requirement of cDNA library construction .
The specific primers were designed based on the sequence of the BSL marker obtained by RAPD . About 1kb lengths of digoxigenin-labeled DNA probes were used to detect BSL gene by dot hybridization. We screened Norin 8 rice about library 300,000 cDNA clones by plaque hybridization using probes. Some Positive clones were obtained. Meanwhile, the insert fragments were cloned and sequences were analyzed.
将约5万粒农林8号m干种子，平均分成12等分，分别用γ射线(0，200，225，250，275，300Gy六个剂量)和离子注入(0，2000，2300，2600，2900，3200六个单位剂量，每个单位剂量约2.6×10~(13)N~+／cm~2)进行诱变处理，处理后的农林8号m发苗至苗二叶期，用0.05％苯达松水培筛选抗性苗，未能获得使农林8号m突变基因功能恢复抗性苗，推测该突变可能不是点突变导致的。
农林8号和农林8号m种子萌发15天左右至苗二叶期。以稻苗为材料，抽提总RNAs，分离mRNAs。分别构建农林8号和农林8号m cDNA文库。初级文库的库容量分别为1.2×10~6，1.4×10~6。随机挑取28个噬菌斑，用T_3、T_7作引物，通过PCR检测，获得插入片断大小从400bp～3kb，平均约1kb。通过x-gal／IPTG蓝白斑鉴定，连接效率99％以上。上述所有皆符合cDNA文库建的总体要求。
参照农林8号和农林8号m RAPD分子标记的结果，设计特异引物，通过PCR获得制备探针的DNA模板。用地高辛标记模板制作探针与农林8号cDNA文库中约30万个克隆子进行Southern原位杂交。获得了一些有杂交信号的噬菌斑，并对目的片断进行克隆和序列分析。
A chemical lethal rice mutant Norin 8 m earring the bentazone sensitive lethal gene was induced from Norin 8 by 60Co gamma-ray radiation. The results indicated that bentazone sensitive lethal gene was controlled by a single recessive gene . The Norin 8 m seedling can be killed by bentazone (above 0.05%), but its FI seedlings are safe. Furthermore, the gene has no negative effects on agronomic traits no matter whether it is homozygous or heterozygous. The techniques for transferring and selecting the marker gene in breeding program and its application ensure the purity of hybrid seed. A making system of the two-line hybrid rice had been put forward.
About 50,000 Norin 8m seeds were divided into 12 parts. The Norin 8m were treated with 0, 200, 225, 250, 275, 300 Gy CO60 and 0, 2000, 2300, 2600, 2900, 3200 X 2. 6X 1013N+/cm2 law energy ion bean implantation respectively. At the 2-leaf stage, all plants were cultived with 0.05% bentazon, the function of mutant gene can't be recovered.
Total RNAs were extracted from seedling of Norin8 and its 60CO gamma-ray irradiation mutant Norin8 m during 2-3 leaves. The messenger RNA was isolated from the total RNA, the Norin 8 and Norin 8 m rice cDNA libraries were constructed. The primary libraies had a total of 1.2 106 phages for Norin 8 and a total 1.4 106 phages for Norin 8 m, 28 randomly selected clones were evaluated. Insert fragments by PCR amplification using T3, T7 primer were obtained and ranged from 400bp to 3kb with an average of 1kb. The 99% phages were recombinants by x-gal/IPTG. All of the above mentioned accorded with the general requirement of cDNA library construction .
The specific primers were designed based on the sequence of the BSL marker obtained by RAPD . About 1kb lengths of digoxigenin-labeled DNA probes were used to detect BSL gene by dot hybridization. We screened Norin 8 rice about library 300,000 cDNA clones by plaque hybridization using probes. Some Positive clones were obtained. Meanwhile, the insert fragments were cloned and sequences were analyzed.
引文
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[2] Lahye T, Shrasn K, Schulze- lefert P.[J] Mol Gen Gent, 1998,29:92-101
[3] 王泽立，戴景瑞，王斌．植物基因的图位克隆[J]．生物技术通报，2000，4：21-27
[4] cause M, et al. [J] Genetics 1994,138:1251-1274
[5] 陈洪，朱立煌，徐吉臣．RAPD标记构建水稻分子连锁图[J]．植物学报，1995，37(9)：677-684
[6] saito A, et al. [j] Japan J Breed, 1991,41:665-670
[7] Auonio B A et al. [J] Rice genetics new Letter, 1993,2(2):3
[8] Xiao J H et al.[J] Rice Genetics New Letter, 1992,9:124-128
[9] Yoshiadi Harushimm, Masahiro Yana Agahiko Shomura, et al.[J] Genetics, 1998,148:479-494
[10] 朱立煌，徐吉臣，陈英．用分子标记定位一个未知的抗稻瘟病基因[J]．中国科学(B)，1994，21(10)：1048-1052
[11] Miyamoto, et al. [J] Mol. Plant-microbe Interact, 1996,9(1):60-13
[12] 曾千春，叶华智，朱祯．稻瘟病分子生物学研究进展[J]．生物技术通报，2003，3：1-7
[13] Ronald PC. Albano B et al. [J] Mol. Gen Genet, 1992,236:113-120
[14] 戳文学，朱立煌．水稻白叶枯病抗性基因的研究与分子育种[J]．生物工程进展，1999，19(6)：9-15
[15] Ray J D.[J] Thor Appl Genet, 1996,92:627-636
[16] wu P, Luo a, Zhu J, et al. [J] Plant and Soil, 1997,196:317-320
[17] 李子银，林兴华，谢岳峰．利用分子标记定位农垦58S的光敏核不育基因[J]．植物学报，1999，41(7)：731-735
[18] Zhang G, Bharaj TS, Lu Y, et al. Mapping of the Rf3 nuclear Fertility restoring gene for W A cytoplasmic male sterility in rice using RAPD and RFLP marker[J]. TAG, 1997,94:27-33
[19] 袁隆平．杂交水稻的育种战略设想，杂交水稻．1987(1)：1-3
[20] 张集文，运用除草剂技术进行杂交水稻除杂保纯的研究．杂草科学2001(2)：2-5
[21] Miyamoto, et al Plant-microbe Interact, 1996(9)1:6-13
[22] Ray J D.[J] Thor Apple Genet, 1996, 92:627-636
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