籼型抗病虫抗除草剂转基因水稻新品系的培育

发布时间：2018-01-25 00:34

本文关键词： 籼型转基因水稻回交育种抗病抗虫抗除草剂　出处：《扬州大学》2017年硕士论文　论文类型：学位论文

【摘要】：水稻是世界上最重要的粮食作物之一,生产安全受诸多因素的影响,其中受病虫草害侵袭最为严重。理想的水稻品种不仅要求高产优质而且还要多抗。因此,培育出可供生产上利用综合性状优良的多抗转基因水稻新品种(系),可以为粮食生产安全提供更多的保障,同时还可以减少化学农药的使用、降低生产成本、保护环境和生态平衡。由于传统育种的种种限制因素使水稻抗病虫育种工作不能满足生产需要,而转基因技术的日渐成熟可以使得这些问题得以解决。本研究在前期已获得的多种类型粳稻多抗转基因水稻的基础上,通过回交转育的手段,把抗病基因AP1、抗虫基因Bt和GNA及抗除草剂基因Bar导入到多个籼稻品种中,并选育出不同基因组合的籼型转基因水稻株系。对选育出的转基因水稻株系进行白叶枯病、纹枯病、除草剂和螟虫的抗性鉴定试验,所得主要实验结果如下:1、通过回交转育获得纯合的转基因水稻:本实验室在前期已经获得粳稻背景的多种类型(AP1、Bt、GNA、Br)的转基因水稻,本研究利用已有的转基因株系与籼稻品种杂交,再通过回交转育的途径将多个抗性基因导入到籼稻品种中:将GNA和Bar基因导入到9311中,将AP1基因导入到9311中,将Bt基因导入Ⅱ-32中,将AP1、Bt、GNA、Bar四个基因聚合到中恢8006中,得到4种籼型水稻转基因株系,经过多代的回交再自交,在自交后代群体中筛选出农艺性状与轮回亲本基本一致的籼型转基因水稻株系。2、RT-PCR结果表明目的基因在籼型转基因株系中成功表达:以籼型转基因株系反转录后的cDNA为模板进行目的基因的扩增,都成功扩增出目的条带,证明了目的基因都已导入受体亲本中且成功表达了。3、转基因水稻对病虫害和除草剂的抗性均有提升:抗性鉴定试验结果表明,选育出的转基因水稻与未转化对照相比抗性有不同程度的提高。含有AP1基因的转基因水稻对白叶枯病菌的抗性等级与对照相比有明显提升,达到抗病级别,而亲本的抗病等级表现为中感;对纹枯病的抗性等级虽然同对照一样都是中抗,但亲本9311和中恢8006的病情分级分别是4.25和4.51,而9311(AP1)转基因株系和中恢8006(GNA+Bar、Bt、AP1)转基因株系的病级分别是3.78和4.04,转基因株系与亲本之间的病级有极显著性差异。可推断转基因水稻对纹枯病的抗性有所提高。4、在田间不施农药条件下的抗虫鉴定实验中含有Bt基因转基因水稻株系对螟虫的综合抗性方面表现良好:亲本Ⅱ-32和中恢8006的卷叶率都达到100%,枯心率分别为1.7%和1%;Ⅱ-32(Bt)转基因水稻株系和中恢8006(AP1、Bt、GNA+ Bar)转基因株系的卷叶率分别是3.7%和2.3%,枯心率都为0。可以明显看出Bt基因的转入提高了水稻对稻纵卷叶螟和三化螟的抗性。对除草剂的抗性鉴定试验中含有Bar基因的转基因株系也表现出对Basta的良好抗性。5、转基因水稻的农艺性状与未转化对照相似:对转基因水稻和未转化对照的农艺性状进行调查和分析,结果表明经过回交育种结合农艺性状定向选择选育出的9311(AP1)、9311(GNA、Bar)、Ⅱ-32(Bt)转基因水稻的农艺性状与其对应亲本9311和Ⅱ-32没有显著差异,中恢8006(AP1、Bt、GNA、Bar)多抗转基因水稻除了结实率比对照中恢8006升高外其他农艺性状与对照相比也没有显著性差异。
[Abstract]:Rice is one of the most important crops in the world, the production safety is affected by many factors, by which the pest invasion is the most serious. Ideal rice variety requires not only high quality but also multiple resistance. Therefore, cultivated for the production of the comprehensive characters of new resistant transgenic rice varieties (lines), can be excellent to provide more protection for the safety of grain production, but also can reduce the use of chemical pesticides, reduce production costs, protect the environment and ecological balance. Because of the limitations of conventional breeding to rice disease and insect resistance breeding work can not meet the need of the production and maturation of the transgenic technology can make these problems can be solved. This study has been based in Early Japonica rice variety DUOKANG, through backcross method, the resistance genes AP1, Bt and GNA insect resistant gene and herbicide Agent gene Bar into a plurality of indica rice varieties, and selected different gene combinations of indica transgenic rice. Leaf blight, breeding of transgenic rice lines with sheath blight, resistance identification test of herbicide and borers, the main results are as follows: 1, through backcrossing to obtain transgenic rice homozygous: the lab has access to a variety of types of Japonica background in the early stage (AP1, Bt, GNA, Br) of the transgenic rice, this study uses the existing transgenic lines and hybrid indica rice varieties, and then through the way of backcross multiple resistance genes into indica rice varieties: GNA and Bar gene into the 9311, the AP1 gene into the 9311, the introduction of Bt gene into -32 II, AP1, Bt, GNA, Bar four to restore 8006 gene pyramiding, 4 indica rice transgenic lines, through many generations of backcross and selfing progenies in. The group selected indica transgenic rice lines.2 agronomic traits and the recurrent parent is consistent, RT-PCR results show that the successful expression of target gene in indica transgenic lines in indica transgenic lines after reverse transcription of cDNA as template to amplify target genes, were successfully amplified bands, proved the target gene has been introduced into the receptor parent and expression of.3 transgenic rice, resistance to diseases and insect pests and herbicides were improved: identification of resistance test results showed that the transgenic rice bred and untransformed compared to control resistance is increased in various degrees. The resistance level of AP1 gene containing transgenic rice bacterial blight. Has improved significantly compared with the control, and reached the resistance level, parental resistance grade is in the sense of resistance to sheath blight; although the same level control are the same as in anti, but parents and 9311 In the 8006 grade of disease recovery were 4.25 and 4.51, and 9311 (AP1) transgenic lines and 8006 lines (GNA+Bar, Bt, AP1) disease level of transgenic lines were 3.78 and 4.04, the disease level between transgenic lines and their parents have significant difference. It can be inferred that the resistance of transgenic rice Rhizoctonia disease increased.4 expression in the field application of insect resistant identification experiment under the condition of pesticide containing Bt gene transgenic rice lines resistant to borers comprehensive good parents: II -32 and restore 8006 leaf roll rate reached 100%, 1.7% and 1% respectively for the dead heart rate; -32 II (Bt) the transgenic rice lines and 8006 lines (AP1, Bt, GNA+, Bar) leaf of transgenic lines were 3.7% and 2.3%, the dead heart rate is 0. it is clear that Bt gene increased the resistance of rice to rice Leaffolder and rice. Identification of resistance to herbicide containing test Bar gene The transgenic lines also showed good resistance to.5 Basta, the agronomic traits of transgenic rice and non transformed controls similar: investigation and analysis of transgenic rice and untransformed agronomic traits. The results showed that after backcross breeding combined with agronomic traits of the 9311 directional selection (AP1), 9311 (GNA, Bar). -32 II (Bt) agronomic traits of transgenic rice and its parents 9311 and -32 II had no significant difference in the recovery of 8006 (AP1, Bt, GNA, Bar) in addition to resistant transgenic rice seed setting rate of 8006 compared with the control in recovery increased other agronomic traits compared with the control, there were no significant differences.

【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S511.21

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