摘要
苏云金芽胞杆菌(Bt)产生的品体蛋白对鳞翅目昆虫具有特异的毒性,它作为生物杀虫剂在农业害虫防治中已经应用了几十年。随着植物基因工程的发展,编码这种杀虫晶体蛋白的Bt基因被转入许多作物中用于害虫防治。转基因的Bt作物在1996年开始商业化种植,给目标昆虫群体带来了巨大的选择压,因此面临着昆虫可能对其进化出抗性的风险。为了延缓昆虫抗性的发展,人们设计了很多昆虫抗性治理(IRM)的策略,基因聚合是其中的一种。
水稻是最重要的粮食作物之一,全世界一半以上的人口以之为主食。虫害是世界范围内水稻生产的一个严重威胁,每年都造成巨大的经济损失。为了防治鳞翅目的昆虫,许多Bt基因被转入水稻中,成功地获得了具有抗虫性的转基因株系。根癌农杆菌介导的转化法是培育Bt水稻最常用的方法,但是转化的过程会引起许多体细胞变异,影响了转基因水稻植株的农艺性状。
本研究以分别转入了cry1Ab、cry1Ac、cry1C和cry2A的4个单价Bt水稻株系为亲本,通过有性杂交的方法将不同的Bt基因聚合起来,培育双价Bt抗虫水稻,并对它们的抗虫性和农艺性状进行了评价。同时,我们还以原品种为轮回亲本,将其中分别转入了cry1Ac、cry1C和cry2A的3个单价Bt水稻株系连续回交3次,最后考察了BCnF3 (n= 1,2,3)纯合株系的农艺性状。本研究获得的主要结果如下：
1.通过PCR方法在F2代检测Bt基因的分离比,证明Bt基因在聚合后代中仍然按照孟德尔规律稳定地遗传,并且在F3代筛选获得了各种组合的双价Bt纯合的水稻株系。
2.酶联免疫吸附测定(ELISA)的结果表明cry1Ab、cry1Ac和cry2A在聚合之后能够稳定地表达,对应的Bt蛋白在双价株系中的含量基本上维持在单价亲本的水平。Cry1C蛋白在双价株系中的含量在聚合之后普遍降低了。Cry1Ab、Cry1C和Cry2A蛋白在不同组织中的含量由高到低的顺序依次为叶片、胚乳、茎杆和根,而Cry1Ac蛋白含量由高到低的顺序依次为叶片、茎杆、根和胚乳。
3.所有转基因株系在室内抗性测验中对一龄三化螟(Scirpophaga incertulas)的致死率为100%,对二龄三化螟的致死率也都高于75%。1Ab+1C、1Ac+1C和1Ac+2A这3种组合对二化螟(Chilo suppressalis)的致死率超过80%,其中1Ac+1C和1Ac+2A产生的致死率显著高于它们的单价亲本,体现出明显的增效作用。另外,二化螟幼虫对Bt蛋白的敏感性显著弱于三化螟幼虫,相同昆虫的二龄幼虫对Bt蛋白的敏感性显著弱于一龄幼虫。
4.在田间的抗性试验中,原品种明恢63(MH63)和汕优63(SY63)受到严重的危害,而所有的单价和双价纯合株系以及杂交种都对钻心虫表现出优良的抗性,出现的枯心极少。转cry2A单价株系及其对应的杂交种受到很轻微的卷叶螟危害,而其它所有的转基因株系和杂交种都完全没有卷叶。
5.单价和双价纯合株系的形态学性状和产量性状出现了一些变异,但是大部分双价纯合株系的单株产量达到了MH63的水平。Bt杂交种农艺性状的变异相对亲本较少,所有双价杂交种的单株产量与SY63相比都没有显著的差异。
6.连续回交和自交后通过发芽试验筛选到了BCnF3纯合株系,它们仍然对水稻鳞翅目害虫保持着优良的抗性。
7.3个单价亲本株系农艺性状的变异在很大程度上得到了恢复,BC3F3纯合株系的单株产量都达到了MH63的水平。
8.对带有cry1C和cry2A的单价亲本株系转基因插入位点进行了详细的分析,通过Southern杂交发现cry1C和cry2A在插入位点上分别有两个和一个拷贝。用反向PCR方法分离克隆了侧翼序列,分别定位在水稻的第11和12号染色体上。
The bacterium Bacillus thuringiensis (Bt) can produce crystal proteins that have specific toxicity to lepidopteran insects, and has been applied as a microbial insecticide to the control of crop pests for several decades. With the development of plant genetic engineering, Bt genes encoding insecticidal crystal protein have been introduced into many crop species for pest controlling. Transgenic Bt crops were first commercialized in 1996 and placed a large selection pressure on the target insect populations, so Bt crops face a risk that insect could evolve resistance to them rapidly. Several insect resistance management (IRM) tactics, including gene stacking, have been proposed to delay the development of pest resistance to Bt crops.
Rice is one of the most important food crops, and the staple food for more than half of the people in the world. Insect damage is a great threat to rice production worldwide, causing serious loss every year. For controlling the lepidopteran stemborers and leaffolders, many Bt genes have been introduced into rice, and transgenic lines with resistance were obtained successfully. Agrobacterium-mediated transformation is the most common method of developing Bt rice, but its transgenic procedure causes somaclonal variation continually and influences the agronomic performance of transgenic rice plants.
In this study, we pyramided two Bt genes to develop two-toxin rice by sexual crossing between four single-gene Bt lines possessing crylAb, crylAc, cry1C and cry2A respectively, and evaluated their insect-resistance and agronomic traits. Meanwhile, we continuously backcrossed three single-gene Bt lines possessing cry1Ac, crylC and cry2A respectively for three times, using their original variety as the recurrent parent, and investigated the agronomic traits of BCF3(n-1,2,3) homozygous lines at last. The main results in this study are as follows:
1. The segregation ratios of Bt gene in the F2 generation were detected through PCR analysis, and it was confirmed that inheritance of Bt gene still follows Mendelian segregation in the pyramided progeny. Homozygous rice lines of two pyramided Bt genes were obtained in the F3 generation.
2. The results of enzyme-linked immunosorbent assay (ELISA) indicated that the expression of crylAb, cry1Ac and cry2A was comparatively stable after gene stacking, and most pyramided lines contained a comparable Bt protein level to their single-gene parents. The expression of cry1C decreased in two-gene lines. The levels of crylAb, cry1C and cry2A expressed in different tissues in a decreasing order was leaf, endosperm, stem and root, while the decreasing order of cry1Ac was leaf, stem, root and endosperm.
3. In the laboratory assay, all transgenic rice lines caused 100% mortality of the first-instar yellow stem borer (Scirpophaga incertulas), and the mortalities of second-instar larvae were higher than 75%. When using striped stem borer (Chilo suppressalis) as test insect, the combinations of lAb+1C, 1Ac+1C and 1Ac+2A caused mortality higher than 80%. Two-gene lines of 1Ac+1C and 1Ac+2A caused apparently higher mortality than corresponding single-gene parents, so the synergistic effects were observed. In addition, larvae of striped stem borer showed higher tolerance to Bt protein compared to yellow stem borer, and the second-instar larvae were less susceptive than the first-instar larvae.
4. In the field evaluation, original varieties Minghui 63 (MH63) and Shanyou 63 (SY63) were damaged seriously. All transgenic rice lines and their hybrids exhibited excellent efficacy against stemborers, with few deadhearts. Single-gene line 2A and its hybrid were damaged slightly by leaffolders, while all the other transgenic lines and hybrids had no fold leaves.
5. Both single and two-gene rice lines showed some variations in morphological and yield traits, but most two-gene lines had a comparable yield per plant with MH63. Bt hybrids showed relatively fewer variations in agronomic traits, and none of the two-gene hybrids had significantly lower yields than SY63.
6. BCnF3 homozygous rice lines were obtained by a germination assay after continuous backcrossing and selfing, and proved to remain resistant to lepidopteran pests.
7. The agronomic traits of three single-gene lines were recovered to a large extent, and the yield per plant of BC3F3 homozygous lines was comparable with that of MH63.
8. The transgene insert sites in single-gene parental lines possessing cry1C and cry2A were analyzed detailedly. Results of Southern hybridization indicated that cry1C and cry2A had two and single copy at the single site respectively. The flanking sequences of cry1C and cry2A were cloned by inverse PCR, and located on chromosome 11 and 12 respectively.
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