【作者】 杨超；

【导师】 卢宝荣；

【作者基本信息】 复旦大学 ， 植物学， 2012， 博士

【摘要】 在过去的半个世纪,粮食产量有了较大的提高,但世界仍有七分之一以上的人口不能获得足够的蛋白质和能量,或处于营养不良状态。转基因生物技术的应用和推广将为全球粮食安全问题提供了新的解决办法。我国抗虫转基因栽培稻的研发国际领先,目前许多抗虫转基因栽培稻品系已经进入环境释放和生产性实验阶段,预示着抗虫转基因栽培稻可能会进行商业化种植,并为我国的粮食安全提供有力保障。抗虫转基因栽培稻的商业化种植面临着两个重要的环境生物安全问题：(1)长期种植抗虫转基因水稻可能使靶标害虫产生对Bt基因的抗性：(2)抗虫转基因通过基因漂移逃逸到野生近缘种将导致潜在的生态影响。在我国栽培稻的主要产区,转Bt抗虫转基因栽培稻的靶标害虫之一为二化螟,其抗性的产生速率将直接影响到转Bt抗虫转基因栽培稻的使用年限及抗虫转基因资源的丧失。同时,栽培稻的种植区内分布着杂草稻和普通野生稻等栽培稻的野生近缘种,随着抗虫转基因栽培稻的商业化种植,抗虫转基因的逃逸将改变野生近缘种种群的适合度和基因传递模式,导致潜在的生态影响。因此,基于上述环境生物安全的基本问题,围绕抗虫转基因(Bt)的靶标害虫二化螟与栽培稻、杂草稻和野生稻之间的相互关系,利用进化生物学的理论预测二化螟对抗虫转基因产生抗性的潜力和抗虫转基因栽培稻的杂交-渐渗对栽培稻、杂草稻和野生稻适合度以及进化潜力的影响。基于以上科学问题,本论文将进行以下几方面的研究：(1)利用荧光AFLP分子标记分析中国二化螟种群的遗传多样性、遗传结构和基因流,以此结果预测抗虫转基因商业化种植后,二化螟对抗虫转基因产生抗性的潜力；(2)利用人工“罩笼法”和接种虫卵的方法,分析在二化螟虫压下栽培稻、杂草稻和普通野生稻适合度的差异,在不涉及转基因材料的条件下模拟抗虫转基因对栽培稻、杂草稻和普通野生稻适合度的影响,并以此预测抗虫转基因逃逸到杂草稻和普通野生稻带来的生态影响；(3)利用共显性分子标记(SSR),检测在栽培稻-杂草稻杂种分离F3及F4后代群体中来自双亲等位基因的分离规律,分析转基因对双亲基因传递的影响,以及作物基因的传递对转基因渐渗的影响。通过上述研究获得以下主要结论：(1)中国东南部地区的二化螟有着中等程度的遗传多样性,较高的基因流导致有限的种群间遗传分化。AFLP分析获得的遗传多样性结果为PLP：59.90%～91.58%,H：0.17～0.27和Ht=0.27。通过F统计,寄生在茭白的二化螟种群与寄生在栽培稻的二化螟种群有着明显的种群分化,FST值为0.275,而12个栽培稻二化螟种群间的遗传分化程度相对较小, FST值为0.120。通过AMOVA分析,有88%的遗传变异划分在种群内部,只有12%的变异存在于种群之间或者不同的稻区之间。对12个以栽培稻为寄主的二化螟种群进行Mantel检测发现,种群间遗传距离和地理距离不存在显著相关关系(r2=0.024；P=0.118；置换999次)。利用间接方法估算13个二化螟种群间的基因流,基因流的估算值Nm约为1.63；去除茭白二化螟种群,基因流的估算值Nm约为2.62。基于个体的指派检测显示大部分的个体(56%)被指派到样本采集时所在地的种群,有19%的个体被指派到其他采集地的种群,还有约25%的个体被指派到本研究中未涉及(没有采集的区域)的种群中。因此推断,二化螟种群间存在较高水平的基因流会延缓二化螟对Bt基因抗性的产生。(2)连续三年的人工“罩笼法”隔离和接种二化螟虫卵的比较实验,结果表明：抗虫转基因逃逸后可能会为杂草稻带来较大的适合度利益,但是为野生稻带来的适合度利益非常有限,以此判断对野生稻的生态影响较小。在2009年、2010年和2011年人工罩笼和接种二化螟的实验中,接虫笼内栽培稻的枯心率分别是17.9%、18.4%和5.4%,而在不接虫的罩笼内栽培稻种群上的枯心率分别为2.7%、4.1%和0.5%。在二化螟虫压较大的2009年和2010年,栽培稻和杂草稻的株高、分蘖、穗数、单株小花数、单株种子数和结实率在接虫罩笼内均显著低于不接虫罩笼内。而在虫压较小的2011年,栽培稻的单株种子数在接虫罩笼内仍然显著低于不接虫罩笼。连续三年的实验中,普通野生稻生殖生长时期的大部分适合度性状在不同接虫处理间未检测出显著性的差异,说明普通野生稻对二化螟有一定的补偿效应或者表现出耐虫性,可以预测抗虫转基因的逃逸对普通野生稻适合度的影响较小。以上的结果与前人关于抗虫转基因对栽培稻、杂草稻和普通野生稻适合度效应影响的研究结果基本吻合,说明人工“罩笼法”处理方式可以有效的模拟抗虫转基因逃逸对栽培稻、杂草稻和野生稻种群带来的生态影响。(3)在抗虫转基因栽培稻-杂草稻的杂种分离F3和F4代种群中,有大量的位点(52%-88%)发生了非随机分离,水稻的自交特性和环境选择可能是有较多位点发生非随机分离的主要原因。在发生非随机分离的位点,并没有明显的偏向于父本或母本,说明双亲基因都可以成功的传递到高世代的群体中。以卡方检测分析不同世代间基因频率的变化,结果表明含有抗虫转基因的组合中近一半(48%)的位点的基因频率在世代间没有显著性差异,而不含有抗虫转基因的组合中只有20%的位点的基因频率在世代间没有显著性差异,说明抗虫转基因可能通过适合度的改变降低了含有转基因的组合所受到的选择压力,而对世代间的基因传递产生了影响。综上所述,研究发现中国东南方稻区的二化螟种群存在中等程度的遗传多样性和较高水平的基因流,产生抗性的可能性相对较低,配合以合理的“高剂量-庇护所”可以更有效延缓抗性的产生。此外通过人工“罩笼法”和接种虫卵方法对抗虫转基因作用的模拟,可以推断抗虫转基因的逃逸将对杂草稻和栽培稻的适合度产生较大影响,而普通野生稻通过补偿效应弥补了二化螟的取食危害,使得抗虫转基因对普通野生稻的适合度影响较小。同时不涉及转基因材料的人工“罩笼”法为利评价转基因逃逸的生态影响提供了新的手段。在适合度受抗虫转基因影响较大的抗虫转基因栽培稻-杂草稻杂种分离群体,在高世代中来自于双亲的基因都可以成功的传递,并且抗虫转基因对世代间的基因传递产生了影响。更多还原

【Abstract】 Remarkable increase in food production has been achieved worldwide over the past half century, but more than one seventh of globle population is under shortage of protein and energy or malnutrition situation. The application and promotion of genetically modified (GM) crops may be a promising solution to food security problem and the insect-resistance GM crops is one of the most widely used and successful transgenic crop. Till 2011, the global planting area of insect-resistance GM crop has reached 24 million hectares, about 15% of all GM crops. Many insect-resistance transgenic rice varieties have access to the stage of national biosafety assessments, promising the future of wide commercial application of GM rice. However, two environmental biosafety concerns are now the key constraints to the future successful commercial application of GM rice:(1) the resistance of target pest; (2) the ecological risk of insect-resistance gene escape.In rice planting area in China, the major target pest of insect-resistance rice is rice striped stem borer (RSSB), whose resistance to Bt toxin will directly threat the longevity of the commercial product. In the meantime, wild relatives of cultivated rice, including weedy rice and common wild rice, are also widely distributed in rice planting areas in China. With the commercialization of GM rice, once the insect-resistance transgene escapes into the wild relative populations, it will alter individual fitness and change the genome structure, which may finally cause a serious biosafety concern. Thus, this research was based on the relation and mutual influence of RSSB and cultivated rice, weedy rice and common wild rice, studying the resistance evolution potential to the Bt toxin by the pests, the impacts of insect-resistance GM rice hybridization-introgression effect to weedy rice and common wild rice.The main research contents include:(1) using fluorescent AFLP (amplified fragment length polymorphism) to estimate genetic variation and gene flow of RSSB populations collected from the Southeast China, which may predict the potential resistance development in rice striped stem borer populations; (2) using nylon cage isolation and egg massive incubation method to detect the tolerance differences between cultivated rice, weedy rice and common wild rice, which may simulate the consequence of insect-resistance transgene escape to cultivated rice, weedy rice and common wild rice population; (3) using the SSR neutral molecular markers to test the distortion segregation in hybrids of insect-resistance GM rice and weedy rice. The main results and findings were exhibited below:(1) The moderate level of genetic diversity combined with relatively frequent gene flow among RSSB populations was detected across Southeast China. The genetic diversity was estimated based on fluorescent AFLP fragments and the results showed that the striped stem borer collected from Southeast exhibited moderate level of genetic diversity (PIP:59.90%～91.58%, H:0.1651～0.2699 and H(=0.27). The limited divergence among populations was detected in RSSB populations collected from Southeast China. Evident genetic divergence was found between the JX stem borer population sampled from water-oat and RSSB populations with the FST value of 0.275. However, a relatively low level of genetic divergence was detected among the 12 RSSB populations, with the overall FST value of 0.120-about 12% of among-population genetic variation was found in the 12 RSSB populations. AMOVA test results showed that majority of the genetic variation was present within populations (>88%), and only a very small proportion (<12%) of genetic variation was found among populations and between the two regions, agreeing with the FST results. In addition, the Mantel test between genetic distance and geographical distance of the 12 RSSB populations did not show pattern of isolation by distance (r2=0.024; P=0.118; and 999 permutations).The level of gene flow among the RSSB populations collected from Southeast China was relatively high, regardless of their geographical distances. The results showed that the average Nm value of all 13 striped stem borer populations was 1.63, and the average Nm value of all 12 RSSB populations was 2.62. In assignment test, about 56% of individuals was assigned into the 12 native populations with a high confidence (P<0.01). However, about 19% of individuals were assigned to neighboring populations sampled in this study, and about 25% of individuals were assigned to other populations that were not sampled in this study. Thus, the results of assignment test also confirmed the considerable level of gene flow between the RSSB populations.(2) After the three-years experiment of nylon cage isolation and egg massive incubation of RSSB, the result show that the insect-resistance transgene escape event greatly enhance the fitness of cultivated rice and weedy rice, contrasting to the low fitness benefit to common wild rice. The artificial nylon cage isolation and egg massive incubation of RSSB were successful:From 2009 to 2011, the white-headed rate of cultivated rice were 17.9%,18.4% and 5.4% respectively in high pest pressure cage, whereas the white-headed rate of cultivated rice were 2.7%,4.1% and 0.5% respectively in low pest pressure cage. In high pest pressure treatment, the cultivated rice and weedy rice had poor performance in plant height, number of seeds per plant, number of florets per plant, grain weight and number of effective panicles compared with the low pest pressure treatment, but the common wild rice had no significant difference. These results indicate that the insect-resistance transgene escape event will greatly enhance the fitness of cultivated rice and weedy rice, contrasting to the low fitness benefit to common wild rice. The results above agree with the previous study in consequence of insect-resistance transgene escape to cultivated rice, weedy rice and common wild rice population, indicating that nylon cage method is able to effectively simulate the consequence of insect-resistance transgene escape to cultivated rice, weedy rice and common wild rice population.(3) In F3 and F4 generations of insect-resistance cultivated rice ×weedy rice, lots of loci (transgene negative combinations:88.89%, transgene positive combinations:66.67%) showed distortion segregation. The inbreed character and environment selection might be the main reason. For those loci, no apparent bias to crop allele or weedy rice allel is detected, indicating that both alleles from parents can be submitted to higher generations. X2 test for the change of gene frequency between different generations showed that lots of loci (48%) in transgene positive combinations remains unchanged while less loci (20%) in transgene negative loci remains unchanged, indicating anti-insect transgene might have influence on the gene trasmition between generations. In F3 and F4 generations, lots of loci showed the pattern of heterozygote excess, which indicates heterozygotes genotype might have advantage in natural selection.In summary, this study found moderate level of genetic diversity and high level of gene flow among RSSB populations in Southeast China, indicating the proper High dose-refuge strategy may slow down the resistance evolution of the RSSB populations. Besides, by the means of nylon cage isolation and egg massive incubation to simulate the effect of transgene, we found the anti-insect transgene escape will influence weedy rice and cultivated rice in their fitness, while common wild rice suffers less from anti-insect transgene for its compensation effect to RSSB. This cage isolation method using non-transgene material provides new means for assessment of ecological consequence of transgene escape. In the hybrid offerings of weedy rice and sensitive cultivated rice to the insect-resistance transgene, crop alleles can be successfully transmitted and fixed in high generation, in the mean time posing influence on gene transmition pattern between generations.更多还原