【作者】 马炳田；

【导师】 周开达； 朱祯； 李平；

【作者基本信息】 四川农业大学 ， 作物遗传育种学， 2002， 博士

【摘要】 水稻(Oryza sativa L)是重要的粮食作物之一，全世界有近半数的人口以稻米为主食。在我国，水稻年播种面积在3000万公顷以上，其中杂交稻的播种面积占50％左右，达1600多万公顷，部分省市如四川、江西杂交稻占90％以上。虫害是影响水稻高产稳产的重要因素之一。目前常采用化学防治，但效果不理想，还带来环境污染、杀灭天敌和人畜危害等问题。农业技术措施及生物防治也不能有效控制水稻害虫的为害。实践证明，抗虫品种的应用是最为经济有效的办法。常规育种由于受到种质资源稀少和生殖隔离的限制，至今还未解决水稻的抗虫性。植物转基因技术的出现和成熟，为我们解决这个问题提供了一种有效的途径。 本研究通过基因枪或农杆菌转化法将修饰的豇豆胰蛋白酶抑制剂基因(sck、修饰的CryIA(C)毒蛋白基因(sbk)、雪花莲凝集素基因(gna)等转入了优良三系杂交稻恢复系SH527、SH163、保持系D62B、D297B、D702B、两系恢复系W2008、两系不育系612S等亲本材料中，获得196个独立的无性系(T0)。以籼稻为受体时基因枪转化法的绿苗获得率为0.93％，高于农杆菌转化法(0.32％)，但总体上转化籼稻比较困难。为此，我们筛选并建立了一套包括三系、两系亲本在内的易于组培的转基因中间受体系统(SH527、D297、BW2008、612S)，最高绿苗获得率可达2.41％。 分子证据和选择标记基因抗性筛选证明外源基因在转基因植株中能够稳定遗传，有的已传递到第四代(T3)并纯合；目的基因与选择标记基因一般为共整合到受体基因组中，在后代中共遗传，并能正确表达，少数植株发生目的基因丢失现象。通过抗性基因聚合和杂交稻的配组发现外源基因的遗传和表达特性能够通过常规杂交转移到其他材料或杂交稻中。获得抗性基因聚合的转基因植株，但外源基因载体结构相同会导致转基因表达减弱。 田间或室内抗虫性鉴定表明转基因植株抗虫性得到提高。在室内用转sck，sbk基因D62B、W2008、SH527等的蛋白粗提物浸泡的饲料饲喂玉米螟12天后，平均虫重在15-22mg；未转基因植株蛋白粗提物处理的饲料喂虫后的虫重却有28-30mg，不用植物蛋白粗提物处理的饲料喂虫的虫重达37.60mg。在田间，基因枪转化法获得的转sck基因SH527的感虫分蘖率为15.31％、转sbk基因的为10.54％，对照为35.60％。同样，转sck基因的W2008的感虫分蘖率为24.28％、转sbk基因的为22.29％，对照为40.89％。 转基因的插入和组培过程可能导致受体的一些农艺性状的变异，但绝大多数是不可遗传的，在后代中能够恢复，仅少数为不可逆变异。转gna SH527当代(T0)植株株高仅有66.7cm，而相应的组织培养苗株高为84.0cm，种子实生苗株高为107.3cm；到T1代及T2代，转基因植株的株高与对照相当。转sckW2008的T0代植株在单株分蘖数、株高、穗长、穗粒数、结实率、千粒重等农艺性状上都明显低于未转基因的对照植株。到T1、T2代时，转基因植株与对照植株差异不明显。但转bac(sbk+sck)基因的612S未获一粒种子。 用转Sob基因纯合SH527一个单株作父本与二个不育系（G46A、D62A、D702A）配组的杂交稻的 感虫株率分别为 20％、50％、75％，对照为 80％、85％、80％；感虫分桑率分别为 6．15％、5二8％。 13．70％，对照为18。30％、14．96％、14．13％。考种结果发现转基因杂交稻与相应对照在株高、单株平 均分孽数、平均穗长、结实率、干粒重等主要农艺性状无明显差异。 为了避免选择标记基因给转基因植物带来的担忧，我们首次利用在体外构建的能去除选择标记 基因的“双T－DNA”单子叶植物表达载体系统转化水稻。在转基因植株中，目的基因与选择标记基 因共转移频率为58．旧％：在两个共转移单株的川代中，获得无选择标记基因的转抗虫基因水稻植 株的频率为 20．83O和 18．420。 外源基因在转基因植株中经常发生表达不稳定或沉默现象，严重影响转基因植物的开发与应用。 核基质结合区（MARS）能提高并稳定外源基因在转基因植株中的表达。为此，我们首次利用含豌豆 MARs序列的单子叶植物表达载体转化水稻。在转基因川代中，含MARs的转sob基因W2008、 SH527的日的基因表达活性与不含 MARS的转SCk基因植株相比，分别提高25．25％和 22．40％。含 MARS转基冈＊2008、SN父7个体的表达活性差异分别是最高为最低的2．82倍和4．16倍，相应的不 含 MARS的转基因的为 7．96倍和 11．53倍。更多还原

【Abstract】 ABSTRACT: Rice (Oryza sativa L.) is one of the most important food crops and almost half of the world population depends on it as the major source of nutritional calories. In china, rice is cultivated over 30 million hectares, about 50 percent of which is hybrid rice. Hybrid rice has a distinct yield advantage over inbred varieties, however, is vulnerable to the attack of wide range of destructive insects. To control insects, insecticides are commonly used today. But chemical control is not only ineffective, but also deleterious to human beings, animals and beneficial insects. It is economical and effective to control insects with the insect-resistant varieties, nevertheless which might be bred barely by the traditional methods because of the scarce insect-resistant germplasms. Recent advances in plant biotechnology make it possible to produce insect-resistant rice.Here, we reported that three insecticidal genes (sck: signal-Q?7Y-KDEL; sbk: signaI-CryIA(C)-KDEL; gna: Galanthus nivalis agglutinin gene) were introduced into the elite CMS (Cytoplasmic Male Sterile) restorer lines (SH527, SHI 63), CMS maintainer lines (D62B, D297B, D702B), PTGMS (Photoperiod-Temperature sensitive Genie Male Sterile) restorer line (W2008) and PTGMS (612S) and 198 independent lines were obtained with bombardment or Agrobacterium-mediated transformation. It was difficult to do genetic transformation of different indica varieties. Then, we selected and constructed a set of indica varieties easy to be transformed, consisting of the parents of CMS and PTGMS hybrid rice including SH527, D297B, W2008 and 612S. When the calli from them were used as the target for transformation, the highest percentage of obtaining transgenic plants was 2.41%.It was indicated that the exogenic genes were integrated and inherited in the transgenic plants with molecular analysis and selection by Hygromycin B, but the insect-resistance genes were lost in few transgenic plants. Some homozygous transgenic lines have been already obtained. The nontransgenic plants could get the exogenic genes while hybridized with transgenic plants. The expression of the transgenes would be lowered if their vectors were similar.The resistance to insects of the transgenic plants was improved through genetic engineering. After fed the first instar of corn borer (Ostrinia nubilalis) with the forage treated with the total protein extracts from the transgenic rice plants containing sck and/or sbk for twelve days, the average weight of one larva was from 15 to 22mg, that fed with the forage treated with the total protein extracts from the nontransgenic rice plants was from 28 to 30mg and that fed with the forage untreated with the total protein extracts from rice plants was 37.60mg. In field evaluation, the percentages of damaged tillers of transgenic SH527 plants containing sck or sbk by bombardment were 15.31% or 10.54% and that of the relative check plants was 35.60%. Similarly, the percentage of damaged tillers of transgenic W2008 plants containing sck or sbk by bombardment was 22.29% or 10.54% and that of the relative check plants was 40.89%.The introducing exogenic genes and the tissue culturing courses had some effects on the agronomic traitsof their recipient plants, but most of these variations could not be delivered to their progenies. For example, the plant height of the first progeny of transgenic SH527 plants containing gna was 66.7cm, of nontransgenic plants regenerated from the calli was 84.0cm and of the check plants from seeds was 107.3cm. However, the height of the second or third progeny plants was similar to the check plants. The agronomic traits of the first progeny of transgenic W2008 plants containing sck were influenced by the transgenes, the discrepancies vanished between transgenic and nontransgenic plants by the time of the second or third progeny.The transgenic hybrid lines were produced from hybridization of G46A, D62A and D702A X SH527-$c? In field evaluation, the percentages of the damaged plants of the three hybrid lines were 20%,50% and更多还原