【作者】 王紫萱；

【导师】 易自力； 邓启云； 蒋建雄；

【作者基本信息】 湖南农业大学 ， 植物学， 2004， 硕士

【摘要】 稻瘟病是一种严重危害水稻的真菌病害。D2-1-2(中花9号ZH9(R))是中国科学院遗传与发育生物学研究所以溶菌酶目的基因和npt-Ⅱ选择标记基因作外源基因，用基因枪法转化、经过连续5年田间选育和抗稻瘟病实验获得的T7代转基因水稻纯合品系，抗谱分析表明，该材料对所有参试的37个稻瘟病生理小种均表现出免疫级抗性。针对部分杂交稻在生产上不抗病的问题，本研究以ZH9(R)为溶菌酶基因供体，利用几个目前生产上有广泛应用价值的杂交水稻组合两优培九、汕优63、培两优288和培两优E32的恢复系9311、明恢63(MH63)、288、E32和两优培九、培两优288的不育系培矮64s(PA64s)、进行回交转育，选育出优良的抗病恢复系和不育系，进而配制新的杂交稻组合，以期获得既抗稻瘟病又农艺性状优良的超级杂交稻，以克服杂交稻稻瘟病抗性差的弱点。 (一)带npt-Ⅱ选择标记基因转基因水稻快速检测技术的研究 本研究部分以转基因水稻纯系材料ZH9(R)及受体品种中花9号(ZH9(CK))为材料。以ZH9(R)中携带的npt-Ⅱ基因作为辅助筛选标记，利用抗生素对其进行处理，建立了一套快速检测转基因水稻的技术体系。 通过使用不同浓度的卡那霉素和G418溶液对ZH9(R)和ZH9(CK)成株离体叶片进行处理，当卡那霉素浓度为700mg/l时，到第六天，ZH9(R)和ZH9(CK)二者的叶片都没有受到明显伤害，当G418浓度为80mg/l时，到第四天，ZH9(CK)叶片黄化死亡，而ZH9(R)叶片仍为正常绿色。所以，我们将G418作为筛选转基因水稻的抗生素；将G418(溶液)80mg/l浓度作为筛选转基因水稻成株叶片的临界浓度。同时，我们进一步用G418对ZH9(R)和ZH9(CK)种子、幼胚和幼苗进行了处理，将：①G418(溶液)300mg/l浓度(处理7天)作为筛选转基因水稻种子的临界浓度。②G418(培养基)200mg/l浓度(处理10天)作为筛选转基因水稻幼胚的临界浓度。③G418(培养基)150mg/l浓度(处理12天)作为筛选转基因水稻幼苗的临界浓度。 根据npt-Ⅱ基因和溶菌酶基因序列设计两对引物，对上述筛选方法进行了PCR验证，发现G418阳性株均扩增出了与npt-Ⅱ基因和溶菌酶基因大小相同的带，这验证了G418检测的正确性，同时也说明了npt-Ⅱ基因和溶菌酶基因是紧密连锁的。应用以上确定的临界浓度对转育后代的叶片、种子、幼胚和幼苗进行了筛选，效果都非常明显，经xZ测验，转育回交后代呈1:1分离规律，回交自交后代呈3:1分离规律，符合孟德尔遗传规律，说明外源溶菌酶基因是呈单拷贝、显性、稳定遗传的。(二)zH，卫)转育杂交稻亲本的研究 将双9(R)分别与恢复系9311、MH63、288、E32和不育系PA64s杂交获得Fl代，然后以各亲本9311、MH63、288、E32、PA64s为轮回亲本，进行溶菌酶抗病基因的转育。通过研究得到了如下结论: (1) G418筛选与PcR、PCR一Southem检测的结果是一致的。通过对转育后代进行G418筛选和PCR检测，结果表明以18筛选的阳性株有99%以上扩增出了与溶菌醉基因大小相同的带。为进一步验证目的基因已整合到水稻基因组DNA中，用辣根过氧化物酶标记的目的基因作探针，对部分PcR结果进行了southem杂交，发现PcR阴性株没有杂交信号出现，而阳性株都有明显杂交信号，证实了PCR扩增带确实是溶菌酶基因的特异性片段。(2)将轮回亲本93n、MH63、288、E3么 .PA64s;转育后代93n/zHg(R)、MH63八王9(R)的B几Fl和Bq凡代;288左升9冈、E32龙升9(R)、PA64s/ZHg(R)的BqFI和BCI凡代;杂交组合两优培九、汕优63和培两优288;测交组合BZRA64s旧293n，珍汕97A/B2MH63，扩RA从s旧1288种植于病圈中，以培两优邓8为诱发品种。采取培养稻瘟病菌人工接种和自然诱发相结合，整体发病状况较好。通过发病率和病情指数调查，发现转育后代抗病能力比亲本大大增强，各测交组合的抗病能力与对应的杂交组合相比也有很大的提高。 (3)通过对亲本及各转育后代的株商、穗长、结实率、千粒重和单株产童筹进行农艺性状分析，表明随着回交世代的增加，各转育后代的农艺性状越来越接近于亲本。 (4)为了考查转育后代的杂种优势，本研究对各侧交组合和对照杂交组合进行了农艺性状分析，各测交组合的大部分农艺性状都比对照组合要差一点，这主要是由于低世代回交代(BqFI代或BCIF;代)还不是很纯合，与亲本差异太大的缘故。但部分测交组合有些性状与对照组合还是没有构成显著性差异。更多还原

【Abstract】 Blast is a fungus disease of rice. Using lysozme and npt-II gene as the external genes, transforming by bombardment, by continuous five-year selection and breeding and blast-resistance experiments, Institute of genetics and developmental Biology, Chinese Academy of Sciences obtained Transgenic rice D 2-1-2 (Zhonghua 9 ZH9 (R) ). The analysis showed that it was immune to 37 physiological races of blast. Using the transgenic ZH9 (R) as donor, restorer lines 9311, MH63, 288, E32 and male sterile line PA64s as receptor , restorer lines and male sterile line with lysozme will be developed. Resistant restorer lines crossing male sterile line will obtain good super-hybrid rice that are resistant to blast.(一) Research on quick testing-technology of transgenic rice with npt -II screen marker geneBy using npt- II in ZH9 (R) as auxiliary selection-marker, conducting ZH9 (R) and ZH9 (CK) with antibiotic, we built a system of quickly testing transgenic rice offspring.Detached leaves of ZH9 (R) and ZH9 (CK) were treated by Kanamycin and G418 with different concentration. When Kanamycin was 700 mg/1, on the sixth day, the leaves of ZH9(R)and ZH9(CK)were not damaged. When G418 was 80mg/l, on the fourth day, the leaves of ZH9 (CK) had been yellow and dead, but the leaves of ZH9 (R) remained green. Therefore, G418 was selected as the optimal antibiotic and 80mg/l was treated as the optimal critical concentration to test detached leaves of transgenic rice. Further study indicated: 300mg/l was the best critical concentration to test seeds of transgenic rice; 200mg/L was the best critical concentration to test young embryo of transgenic rice;150mg/Lwas the best critical concentration to test seedlings of transgenic rice.Two primers were designed based on npt- II and iysozme gene sequences. PCR was done to confirm the above detection system. Results showed the wpt-II was closely linked with lysozme gene. Above confirmed critical concentrations were adopted to select detached leaves, seeds, young embryos and seedlings of transferred offspring. Hie effect was very significant. By x testing, the separation law of backcrossed offspring was 1:1 and one of self-crossed offspring was 3:1, which was coincident with Mendelian genetic law. So external lysozme gene was single-copied, dominant and stably genetic.(二) Research on ZH9(R) transferring different hybrid rice parentsZH9(R ) crossed with excellent restorer lines 9311, MH63, 288, E32 and sterile line PA64s, which produced FI generation. Then 9311 , MH63, 288 , E32, PA64s as rotation parents, disease-resistance gene of Lysozme was transferred. The main conclusions are described as follows:(1) G418 testing is consistent with PCR, PCR-Southern testing. Transferring offspring were tested with G418 and PCR. Positive plants of G418 had the same amplifications as one of lysozme gene. Furthermore, PCR conclusion was proved correctly through PCR-Southern. G418 testing is consistent with blast-resistance. 99% of G418 positive plants wete resistant through resistance evaluations.(2) PeiLiangYou 288 as induction variety, rotation parents, transferring generations, hybrid rice and testcrosses were evaluated in rice blast nursery. Combined with inoculation and induction, the effect is satisfactory. The disease-resistance ability of transferring generations was stronger than one of rotation parents. The disease-resistance ability of testcrosses also had been improved greatly compared with corresponding hybrid rice.(3) Investigation on agronomic characters of transferring progenies and rotation parents were made. With the generations increasing, the agronomic characters of backcross progeny are closer with parents. That is to say, all agronomic characters ofBC1F1 have significant differences with rotation parents; some agronomic characters of have significant differences with parents; none of BC3F1 has significant differences with them.(4) Domination of transferring generations was examined. The agronomic characters of testcrosses were worse than one of hybrid rice,更多还原