【作者】 左娇；

【导师】 强胜；

【作者基本信息】 南京农业大学 ， 发育生物学， 2010， 博士

【摘要】 转基因水稻的研发和商品化应用将为我国的水稻生产能力进一步提高提供新的机遇。但是转基因水稻的大规模环境释放和商品化生产可能会带来一定的环境生物安全问题,处理不好就会影响转基因水稻的进一步研究和发展。抗除草剂转基因水稻田间释放带来的生态问题之一,就是抗性基因通过基因流漂移到野生/杂草近缘种中,从而给当地的生物多样性造成威胁。因此,在释放前对其潜在的风险做出评估十分重要。转基因水稻与野生／杂草近缘种间花粉的基因漂移有杂交、回交以及持续这种方式所产生的后代的适合度问题,这些是本文所关注重点。2006至2007年,在温室条件下,对抗除草剂转基因水稻Y0003和99-1与马来西亚杂草稻(WRl)和国内安徽塘稻(WR2)人工授粉获得的携带抗性基因的杂交一代、二代与相应杂草稻回交,得到回交代,统计回交结实率和携带抗性基因的比例,并对携带抗性基因的杂交/回交代的适合度进行了测定,综合判断抗性基因漂移的风险。研究表明,在人工授粉的条件下,携带抗性基因的杂交代都能和相应的杂草稻回交并结实,结实率在15-60%之间。正反杂交一代的种子绝大多数都表现出了良好的抗性,杂交二代和回交代分别表现为3：1和1：1的抗性分离比例,分离比例都符合孟德尔遗传规律。适合度的研究表明杂交代和回交代和相应的杂草稻相比没有明显差异,大多数的杂交种的适合度和回交代的相差不大,个别杂交代的适合度没有回交代的高。以上研究结果表明转基因水稻的抗性基因有可能通过杂交和回交发生漂移。2007年至2009年,在网室和大田通过人工杂交和天然异交等试验来研究转基因水稻和国内15种典型杂草稻间的亲和性和异交率,为揭示产生不同异交率的关键原因。结果表明,转基因水稻和杂草稻间的人工杂交结实率在31.82%-82.68%之间,不同组合间萌发的供体花粉量和进入杂草稻子房的花粉管数量的显著性差异直接导致不同的杂交结实率。粘着到杂草稻柱头上的水稻花粉数量与杂草稻柱头的形态学特征有明显关系。杂交结实率与水稻和杂草稻间的遗传距离显著负相关,遗传距离越大,杂交结实率越低。转基因水稻与杂草稻间天然异交率变化范围为0-6.66%0。株高和穗高对异交率影响很大。水稻Y0003的株高(144.8cm)高于绝大多数杂草稻种群。远高于水稻Y0003的湛江和昆明杂草稻,远低于水稻Y0003的内蒙古和黑龙江杂草稻以及与水稻Y0003高度适中的连云港杂草稻均未得到任何异交结果。与水稻高度相似的广德、金华和芜湖杂草稻的异交率结果最高。穗高对异交率的影响类似。花期重叠由始花时间、终花时间、盛花时间、花期持续时间和相遇批次决定,它是影响天然异交率的关键因素。研究发现,盛花时间、终花时间、花期持续时间以及杂草稻与转基因水稻的相遇批次直接影响两者间的异交率,而穗长和有效分蘖数对其影响不大。大多数杂草稻种群的盛花时间出现在上午8：20-10：20,而异交率第二高的芜湖杂草稻盛花时间在10：20-11：30,与水稻Y0003开花时间从10：00到13：35,其盛花时间为11：10到12：20相比,花时重叠最长达到20分钟。围绕杂草稻的多棵转基因水稻的花期相遇批次与异交率有显著关系。花期相遇批次主要取决于水稻及杂草稻的花期持续时间,异交率最高的广德杂草稻是由于花期最长,与四批水稻花期全部相遇。水稻与不同杂草稻间的遗传距离也影响到杂交结实率。遗传距离代表着亲和性,从本质上是由柱头形态学特征决定的花粉萌发量及进入子房的花粉管数量决定了水稻和杂草稻间不同的异交率。总之,这些因素相辅相成,共同影响了异交率。但是,转基因水稻和杂草稻之间的基因漂移主要由花期同步性决定,其次取决于遗传亲和性和株高的差异性等形态学特征。2009年在网室以抗除草剂转基因水稻(oryza sativa)Y0003和4种杂草稻(O. sativa f. spontanea)金华(JH)、广德(GD)、扬州(YZ)、芜湖(WH)以及两者杂交所得的携带有抗性基因的杂交一代(F1)为研究对象,研究子代与亲本在田间不同栽培方式、混种比例和除草剂处理下的适合度成分,包括株高、分蘖数、茎直径、剑叶长度,剑叶宽,剑叶面积等营养指标；花粉活力、圆锥花序、小穗数/花序、单株饱粒数、单穗饱粒数、自交结实率、穗长、落粒率、百粒重、种子休眠性等生殖指标。结果表明,栽培方式、杂草稻种群及除草剂处理对F1与亲本各指标的差异影响显著。栽培方式对杂交F1的分蘖数、剑叶长度、茎直径等营养指标及圆锥花序、单株饱粒数、落粒数等生殖指标影响显著。喷药使杂交F1的产量降低。混种主要影响了杂交Fl的生殖生长。扬州、广德、金华及芜湖4种杂草稻,除了对杂交F1的株高、剑叶面积、花粉活力、单穗饱粒数及结实率与两亲本的关系无显著影响外,对其它各指标的影响比较显著。在没有选择压(不喷药及单种)时,杂草稻及杂交F1的总适合度均与水稻持平,差异不显著。但是设定选择压(喷药,混种,栽培方式)后,杂交F1的总适合度均显著低于水稻亲本,但是与杂草稻没有显著性差异。混种比例和栽培方式对水稻、杂草稻及杂交F1的休眠性没有任何影响。2009年在大田以抗除草剂转基因水稻Y0003和广德杂草稻及其杂交F1为研究对象,研究这些亲本及Fl在大田条件下不同栽培方式和除草剂选择压下的适合度,测量指标包括株高、分蘖数、茎直径、剑叶面积等营养指标及圆锥花序、小穗数/花序、穗长、单穗饱粒数、单株饱粒数、自交结实率、落粒率、百粒重、花粉活力、种子休眠性等生殖指标。结果表明,江浦大田与牌楼小区试验中,混种比例与栽培方式对GDF1与亲本各指标的差异影响显著。在江浦试验中,混种比例对GD F1与水稻Y0003的分蘖数、穗长、茎直径、圆锥花序、单株饱粒数及百粒重差异影响显著；栽培方式对二者的剑叶面积、小穗数、落粒率及百粒重差异影响显著。而在牌楼小区试验中,混种比例对GD F1与水稻亲本的穗长影响显著；栽培方式对二者的株高、分蘖数、圆锥花序及单株饱粒数影响显著。在喷药情况下,不管以何种栽培方式及混种比例,江浦及牌楼两处GD F1的株高、茎直径、小穗数及百粒重均显著高于水稻亲本,其花粉活力、单株饱粒数、单穗饱粒数及自交结实率等生殖指标均显著低于水稻亲本。随着GD F1在混种中的比重上升,它的剑叶面积均从与亲本无差异到显著高于亲本。两处GD F1的休眠性均远高于水稻Y0003。综上所述,转基因水稻向杂草稻的基因漂移可能存在,但是,不同杂草稻种群的基因漂移频率差异明显,主要受两者间花期相应程度决定,遗传距离决定的亲和性以及株高等形态特征影响到基因漂移频率。尽管杂交后代及回交后代的适合度并未比亲本提高,而且存在适宜的选择压(不同的栽培方式、混种比例及除草剂处理)时,杂交后代的适合度显著低于水稻亲本,但是与杂草稻亲本差异不显著。当然还需要更进一步的研究这些杂交后代及回交后代的生态适合度变化,从而为转基因水稻的环境安全性做出评价。更多还原

【Abstract】 The development and commercialization of transgenic rice with novel traits may promote rice productivity. As rice is a major food crop in China, the enhancement of rice production is important for national food security. If left unaddressed, the potential biosafety issue over the release and commercial cultivation of transgenic rice may hamper the development and application of this technology. One of the ecological concerns over genetically modified herbicide-resistant crops is the gene flow to their wild/weedy relatives, which might result in a menace to local biodiversity. For this reason, it has to be assessed before their release in field. Crop-to-wild/weedy gene flow mediated by pollen involves the efficiency of hybridization, backcross and fitness of their hybrids. These are the focuses of our research.From 2006 to 2007, the F1 and F2 generation hybrids between transgenic herbicide-resistant rice (Y0003 and 99-1) and Malaysia weedy rice(WR1) as well as Anhui weedy rice (WR2) were backcrossed with corresponding weedy rice to obtain backcross generations, and these experiments were performed in greenhouse. We counted the seed setting rates of the backcross generations, tested the proportion of plants carrying the resistance gene in the backcross generations, and observed the fitness of hybrid and backcross generations in order to comprehensively assess the risk of gene flow from herbicide-resistant rice to weedy rice. The results showed that the hybrid generations were able to backcross with corresponding weedy rice and set seed with the range of 15-60% the seed setting rate. The results also indicated that the seed from reciprocal hybrid F1 generation showed favorable herbicide-resistance, but F2 and backcross generations showed 3:1 and 1:1 segregation proportion of resistance respectively, which followed Mendelism genetic law. The study on fitness showed that the fitness of hybrid and backcross generations was not obviously different from that of their corresponding weedy rice. Most hybrid generations were not largely different from backcross generations in fitness, whereas some hybrid generations were lower than backcross generations in fitness. All above results indicate that the herbicide-resistant gene flow from transgenic rice possibly occurs via hybridization and backcrossing.The manual and natural crossing experiments in the network room and field between 2007 and 2009 were conducted to compare the compatibility and outcrossing rates of transgenic rice with those of selected different weedy rice biotypes and to elucidate the key innate factors causing the different outcrossing rates. Hybrid seed sets from manual crossing procedures between transgenic rice and weedy rice varied from 31.82% to 82.68%. The significant differences in the quantity of germinated donor pollens and pollen tubes entering the weedy rice ovule directly contributed to the different seed sets. The number of transgenic pollen grains adhering to the stigma of weedy rice biotypes was related to the morphological characteristics of their stigma. Results showed that seed sets of manual crosses were negatively correlated to the genetic distances between Y0003 and the different weedy rice biotypes.The natural outcrossing rates varied from 0 to 6.66%o. Plant and panicle height affected outcrossing result. Results showed that the height of transgenic rice Y0003 (144.8cm) is just larger than all the weedy rice biotypes. The weedy rice biotypes much higher than rice Y0003 (Zhanjiang and Kunming) and the ones much lower than rice Y0003 (Inner Mongolia and Heilongjiang) did not outcross at all with transgenic rice Y0003 unlike the Lianyungang biotype whose plant height was similar to rice Y0003. The Guangde, Jinhua and Wuhu biotypes, which are of a similar height to Y0003, showed the highest outcrossing rates. The panicle height exhibited similar effects on outcrossing rate to plant height.The duration of flowering overlap was the key factor influencing natural outcrossing which was determined by the time of first and final flowering, peak flowering, flowering duration and plant batch of flowering overlap with Y0003. The study showed that peak flowering, time of final flowering, flowering duration, and plant batch of flowering overlap between weedy rice biotypes and Y0003 directly influenced the outcrossing rates. Panicle length and number of effective tillers did not influence the outcrossing rates. The peak flowering of most of the weedy rice biotypes occurred between 08:20am and 10:20am except for Wuhu weedy rice (10:20-11:30). Y0003 rice flowered between 10:00 and 13:35, and its peak flowering time (11:10-12:20) overlapped with that of Wuhu weedy rice, which exhibited the second largest outcrossing rate for about 20 minutes. The weedy rice flowering overlaps with the 4 batches of Y0003 showed a positive relationship with outcrossing rate. Florescence batch of 4 transgenic rice plants around weedy rice was significantly related to the outcrossing rates. Florescence batch of rice and weedy rice depended on their flowering duration. The flowering duration of Guangde weedy rice which exhibited the largest outcrossing rate was the longest, and overlapped with all 4 batches of Y0003.The genetic distance between the various weedy rice biotypes and the transgenic rice affected the seed sets of hybrids. The genetic distance represented the compatibility. And the amount of germinated pollen and the numbers of pollen tubes entering the ovule as determined by morphological characteristics of their stigma essentially determined the different outcrossing rates between transgenic rice and the weedy rice biotypes. All these factors in combination affected the outcrossing rate. While the likelihood of gene flow between transgenic rice and weedy rice biotypes was predominantly determined by floral synchronization and subordinately influenced by genetic compatibility and morphological characteristics such as plant height difference and so on.From 2009, fitness components of parents and F1 hybrids were compared among four types of weedy rice Jinhua (JH), Guangde (GD), Yangzhou (YZ), Wuhu (WH) and herbicide-resistant transgenic rice Y0003 under different cultivation patterns, planting proportions and herbicide treatment. We measured plant height, tiller number/plant, stem diameter, flag leaf length, flag leaf width, flag leaf area in vegetative growth, and pollen viability, panicle number/plant, spikelet number/panicle, filled grain number per panicle, filled grain number per plant, seed set of self-pollination, panicle length, seed shattering, 100-grain weight, seed dormancy in reproduction. The results showed that cultivation pattern, weedy rice populations and herbicide treatment significantly affected the trait differences between F1 hybrids and parents.Cultivation pattern significantly affected tiller number, flag leaf length and stem diameter of F1 hybrids in vegetative growth and panicle number/plant, filled grain number per plant and seed shattering of F1 hybrids in reproductive growth. Spraying herbicide reduced the production of hybrid F1. Mixed planting mainly affected the reproductive growth of F1 hybrid. Biotypes of four weedy rice played significant roles in the relations of F1 and parents in all the variables except plant height, flag leaf area, pollen viability, filled grain number per panicle and seed set of self-pollination. Total fitness of weedy rice and F1 were not significantly differet from transgenic rice Y0003 without select pressure (not spraying herbicide and planting individually). While setting select pressure (herbicide spraying, mixed species and cultivation pattern), hybrid F1 ’s total fitness was significantly lower than rice parents, but had no significant differences from weedy rice parents. Planting proportion and cultivation pattern had no effect on the dormancy of rice, weedy rice and F1 hybrids.From 2009, the fitness of herbicide-resistant transgenic rice Y0003, Guangde weedy rice and their hybrid F1 was compred to assess their survival compatibility under different cultivation and herbicide treatments in the field. We measured plant height, tiller number/plant, stem diameter, and flag leaf area in vegetative growth, and panicle number/plant, spikelet number/panicle, panicle length, filled grain number per panicle, filled grain number per plant, seed set of self-pollination, seed shattering,100-grain weight, pollen viability and seed dormancy in reproduction.The result showed that in Jiangpu field and Pailou plot experiments, planting proportions and cultivation pattern significantly affected the traits of both GD Fi and rice Y0003. In Jiangpu experiment, planting proportion affected tiller number/plant, panicle length, stem diameter, panicle number/plant, filled grain number per plant, and 100-grain weight of GD F1 and rice parent, while cultivation pattern affected flag leaf area, spikelet number/panicle, seed shattering and 100-grain weight of GD F1 significantly. In Pailou plot experiment, planting proportion of GD F1 to rice significantly affected panicle length, and cultivation pattern affected plant height, tiller number/plant, panicle number/plant, and filled grain number per plant significantly. In the case of herbicide spraying, no matter what cultivation pattern or planting proportion were, the values of plant height, stem diameter, spikelet number/panicle and 100-grain weight of GD were significantly higher than those of the rice parents, and the values of pollen viability, filled grain number per panicle, filled grain number per plant and seed set of self-pollination were significantly lower than those of rice parent. With the increase of GD F1 number, its leaf area changed from not differently to significantly larger than the rice parent. Seed dormancy of GD F1 from the two sites was much higher than that of rice Y0003.In summary, gene flow from transgenic rice to weedy rice can happen. However, frequency of gene flow to different weedy rice biotypes were significantly different, which was mainly determined by the degree of floral synchronization. Both compatibility determined by genetic distance and morphological characteristics such as plant height difference affected the gene flow frequency too. Although the fitness of hybrids and backcross was not higher than those of the parents, after setting select pressure (cultivation pattern, mixed species, and spraying herbicide), hybrid F1’s total fitness was significantly lower than those of rice parents, but had no significant difference from those of weedy rice parents. However, further experiments need to be done to address to the change of ecological fitness of hybrids and backcross in order to assess the environmental safety of transgenic rice accurately.更多还原