【作者】 李毓；

【导师】 庄伟建；

【作者基本信息】 福建农林大学 ， 作物遗传育种， 2008， 博士

【摘要】 以载体pSPROK、pCAMBIA1300、pCAMBIA1301及拟南芥基因内含子为材料,通过酶切连接首先构建了一个RNAi工程载体pSC1301-347。然而以pSC1301-347为工具,构建了有关水稻隐花色素基因Cryla、Crylb和Cry2的四个RNAi和两个反义RNA表达载体,分别是pSC1301-347-Cryla、pSC1301-347-Cry2、pSC1301-347-Crylab、pSC1301-347-Crylab2、pSC1301-347-antiCryla和pSC1301-347-anticry2,其中前四个为RNAi表达载体,最后两个为反义RNA表达载体。通过农杆菌介导法,用这些表达载体分别转化粳稻日本晴水稻,得到200多株不同克隆的Gus阳性植株。经测序分析与转基因植株分子检测证明构建的所有表达载体插入序列正确,目的基因片段均已完整地整合于水稻基因组序列。对T₀代转基因植株的观察分析,初步认为:1.隐花色素基因对水稻结实率影响较大从已完成一个生命周期的4个RNAi转基因植株表现来看,植株均能正常抽穗开花,但基本不结实。pSC1301-347-Crylab2（V3）和pSC1301-347-Crylab（V9）未得到一粒种子,结实率为0;pSC1301-347-Cryla（V16）和pSC1301-347-Cry2（V19）个别植株得到少量种子。而对照日本晴结实率可达20%-30%。结实率低的原因,除冬天气候因素和转基因当代植株生长较为异常之外,推测是因为隐花色素基因被敲除或抑制后,水稻幼穗分化、减数分裂、花药开裂等某个或某些代谢过程发生障碍,导致最终不能形成正常有活力的雄蕊。2.子房膨大,不灌浆植株开花后,颖花或者空瘪,或者子房膨大不灌浆,内无淀粉等光合产物积累,只有水状的液态物质。pSC1301-347-Crylab2（V3）和pSC1301-347-Crylab（V9）两个载体的转基因植株尤其如此。推测在两个及两个以上的隐花色素基因表达受到抑制时,淀粉的生物合成或运输途径出现障碍。3.花药不正常雄蕊干瘪,颜色暗淡,花粉粒很少,不散粉。pSC1301-347-Crylab2（V3）和pSC1301-347-Crylab（V9）两个载体的转基因植株表现尤甚。推测cryptochrome基因可能参与水稻幼穗分化的某个生理生化代谢过程。4.植株形态上的差异在福州冬季光照不足的玻璃温室的环境条件下,pSC1301-347-Crylab2（V3）和pSC1301-347-Crylab（V9）转基因植株明显表现出一种病态:株高比pSC1301-347-Cryla（V16）和pSC1301-347-Cry2（V19）稍高,叶色发黄枯萎,叶片似长而软。5.对开花的影响pSC1301-347-Crylab2（V3）和pSC1301-347-Crylab（V9）转基因植株开花时间均比pSC1301-347-Cryla和pSC1301-347-Cry2要迟,因此cry基因与水稻开花有关,且抑制的基因数目越多,对开花似影响越大。以水稻成熟种子和幼嫩种子为材料,对水稻胚性愈伤诱导及其遗传转化进行了研究。结果表明,成熟种子、新鲜成熟种子及幼嫩种子愈伤诱导率均＞81%,平均86.67%,其中以新鲜成熟种子愈伤率最高,达96.887%;抗性愈伤率成熟胚为22.21%-40.71%,平均28.49%,幼胚为36.79%-43.21%,平均40%,幼胚的抗性愈伤率高于成熟胚;分化出苗率成熟胚为59.29%-80.43%,平均71.59%,幼胚为63.16%-72.73%,平均67.95%,幼胚与成熟胚差异不明显;转化率（Gus检测阳性率）成熟胚为43.07%-81.08%,平均61.59%,幼胚为58.33%-76.67%,平均67.50%,幼胚转化率稍高于成熟胚。不同载体抗性愈伤率、分化率与转化率存在一定的差异,表明插入的外源基因片段不同对抗性愈伤率、分化率与转化率有一定的影响。更多还原

【Abstract】 Using vectors pSPROK,pCAMBIA1300,pCAMBIA1301 and an intron from one of Arabidopsis thaliana genes as materials,an engineering vector named pSC1301-347 was constructed.Then,using pSC1301-347 as a tool vector,four RNAi and two antisense RNA expression vectors related to rice cryptochrome genes were constructed.They are pSC1301-347-Cryla,pSC1301-347-Cry2, pSC 1301-347-Crylab,pSC1301-347-Crylab2,pSC1301-347-antiCryl a and pSC1301-347-anticry2 respectively,of which the former four vectors are RNAi ones,and the last two vectors are antisense RNA ones.Through the method of Agrobacterium-mediated transformation,these expression vectors were used to transform japonica rice cultivar "Nipponbare",and finally more than 200 different colonial Gus positive plants were obtained.After sequencing analysis and the molecular assays of transformed plants,the inserted sequences of all the expression vectors were proved to be correct with the target gene fragments completely integrated into rice genome sequences.Based on the observation and analysis of T_o generation transformed plants,the preliminary conclusions were as follows:1)Cryptochrome genes had great effect on the seed-set percentage of ficeAccording to the performance of the four RNAi transformed plants which each had finished one life cirle,all the plants could normally headed and flowered,but basically bore no fruits.Transformed plants from pSC1301-347-Crylab2(V3)and pSC1301-347-Crylab(V9)did not get even one grain of seed,and only one or two plants from pSC1301-347-Cryla(V16)and pSC1301-347-Cry2(V19)bore a small amount of seeds while the seed-set percent for the control variety "Nipponbare" could reach as high as 20-30%.The reasons for the low seed-set percent were probably that besides the cold weather in winter and the abnormity in the growth of the transformed plants,because of the knockdown or restraint of the cryptochrome gene(s),there existed some metabolic disturbances during the rice young panicle differentiation,meiotic division or anther dehiscence and hence the anthers with normal vigor could not be produced finally. 2)Ovaries swelled,but did not filledAfter flowering,the rice spikelets were empty,or only the ovaries swelled with water-like substance in them and no accumulation of photosynthetic products. The transformants from pSC1301-347-Crylab2(V3)and pSC1301-347-Crylab(V9) were especially like this.The reasons for this were probably that when two or more than two cryptochrome genes were restrained in expression,some obstacle(s) occurred during the biosynthesis or transportation for starch.3)Anthers were abnormalAnthers degraded,did not spread microspores with light color and a small number of microspores.The transformants from pSC1301-347-Crylab2(V3)and pSC1301-347-Crylab(V9)were especially like this.The reasons for this were probably that cryptochrome genes were involved in some physiological or biochemical process of rice young panicle differentiation.4)The differences in plant morphologyUder the weak winter sun ligh of Fuzhou greenhouses,the transformants from pSC1301-347-Crylab2(V3)and pSC1301-347-Crylab(V9)distinctly displayed a kind of morbidity.The plants were slightly taller than those from pSC1301-347-Cryla (V16)and pSC1301-347-Cry2(V19),with leaves seemingly longer and softer,and a little whiter and faded.5)The influence on floweringTransformants from pSC1301-347-Crylab2(V3)and pSC1301-347-Crylab(V9) flowered later than those from pSC1301-347-Cryla and pSC1301-347-Cry2. Therefore,cryptochrome genes were related to rice flowering.It seemed that,the more the genes of cryptochrome were restrained,the greater the influence on flowering.Using rice mature and young seeds as material,the studies on rice embryogenic callus induction and the relevant genetic transformation were carried out.The results indicated that the callus induction rate for matue seeds,fresh mature seeds and young seeds was all＞81%,averaged 86.67%,with the induction rate for fresh mature seeds highest up to 96.87%.The resistance callus rate for mature embryo was 22.21%-40.71%,averaged 28.49%,for young embryo was 36.79%-43.21%,averaged 40%,with that for the young embryo higher than that for the mature embryo.The seedling differentiation emergence rate was 59.29%-80.43%with an average of 71.59%for the mature embryo,and 63.16%-72.73%with an average of 67.95%for the young embryo,showing no significant differences between the two kinds of embryos.The transformation rate(Gus positive rate)was 43.07%-81.08%with an average of 61.51%for the mature embryo,and 58.33%-76.67%with an average of 67.50%for the young embryo.The transformation rate for the young embryo was slightly higher than the mature embryo.The resistant callus rate,differentiation rate and transformation rate were different among different vectors to some extent, indicating that different inserted gene fragments had some effects on them.更多还原