【作者】 于恒秀；

【导师】 顾铭洪； 辛世文；

【作者基本信息】 扬州大学 ， 作物遗传育种， 2007， 博士

【摘要】 水稻是世界上最重要的粮食作物之一,全球约二分之一的人口以稻米为主食,传统的遗传育种方法在提高水稻产量、抗性和品质方面已作出了重要贡献,但由于稻属种质资源的限制及常规育种方法的局限性,给水稻进一步的遗传改良带来一定困难。随着分子生物学研究的深入,基因的分离、克隆和重组技术以及转基因技术的日趋成熟,遗传转化已成为水稻遗传改良的一种有效手段。抗虫、抗病和抗除草剂转基因水稻的培育可为水稻的高产与稳产提供重要保证,也可减少化学农药的使用,改善环境质量。通过调控淀粉合成相关基因的表达,可以改良稻米的淀粉品质,以提高其食味品质或加工品质。本研究重点利用转基因技术进行水稻改良的研究,主要研究内容包括两大方面。一是将不同来源的抗虫、抗病和抗除草剂基因,包括苏云金芽孢杆菌(Bacillus thuringiensis,Bt)毒蛋白cryIA(c)基因、雪花莲凝集素(Galanthus nivalis agglutinin, GNA)基因、甜椒编码类铁氧还原双亲蛋白(Amphipathic protein 1, AP1)基因和土壤吸水链霉菌(bialaphos resistance, Bar)抗除草剂基因,导入4个高产粳稻品种广陵香粳、武香粳9号、武香9915和扬辐粳8号中,研究抗病、抗虫和抗除草剂转基因水稻,特别是含有不同目的基因组合的多抗转基因水稻的培育方法,二是,将控制直链淀粉合成的水稻蜡质(Waxy,Wx)基因的不同转基因构建,包括其全长基因组序列(简称全长Wx基因)、Wx-cDNA和反义RNA结构(简称反义Wx基因),导入具有不同直链淀粉含量的水稻品种协青早、龙特甫、武香粳9号、武香9915、苏御糯和广陵香糯等中,研究调控蜡质基因表达对改良稻米品质的效果,在此基础上进一步获得具有优良食味品质或特高直链淀粉含量的水稻新品种(系)。主要研究结果如下:1、转Bt基因水稻。通过双菌双载体或超双元载体介导的农杆菌介导共转化法,将Bt cryIA(c)基因和潮霉素抗性选择标记基因(HPT)同时导入粳稻品种武香粳9号和广陵香粳中,从其自交后代中筛选获得了无HPT基因的转Bt基因水稻材料。抗虫性鉴定结果表明,转Bt基因水稻对稻纵卷叶螟和二化螟的抗性较未转化对照有了明显提高,具体表现为:稻纵卷叶螟对转Bt基因水稻离体叶片的危害程度明显小于未转化对照,转Bt基因水稻离体叶片上的稻纵卷叶螟幼虫全部死亡;二化螟高龄虫对分蘖期转Bt基因水稻离体茎杆危害程度明显小于未转化对照,转Bt基因植株茎杆上只有少量的螟虫排泄物;二化螟幼虫在苗期转Bt基因水稻上死亡率达到100%,较未转化对照有明显提高;二化螟造成的成株期转Bt基因水稻的枯心率明显低于未转化对照,部分转化子对二化螟的抗性由未转化对照的高感上升为抗或中抗级别;在不施用任何农药的田间自然条件下,转Bt基因水稻表现为无稻纵卷叶螟危害,未转化对照的受害率达100%。2、转AP1基因水稻。通过超双元载体介导的农杆菌共转化法,将AP1基因和HPT基因同时导入粳稻品种武香粳9号、广陵香粳中,从其自交后代中筛选获得了含有AP1基因而无HPT基因的水稻。抗病性鉴定结果表明,转AP1基因水稻叶片上白叶枯病病斑长度均极显著小于未转化对照,广陵香粳转基因水稻的抗性级别由未转化对照的中抗上升为抗;转AP1基因水稻虽然与未转化对照对纹枯病的抗性级别均为中感,但相对病班长度均显著或极显著地小于未转化对照;大部分转AP1基因水稻对稻曲病的抗性较未转化对照有所提高。3、转GNA和Bar基因水稻。以粳稻品种广陵香粳、武香9915和扬辐粳8号为材料,以同时含有GNA和Bar基因的双元载体EHA105/pCUGNA-BAR介导的转化法,将GNA和Bar基因导入以上受体品种中,通过除草剂抗性鉴定和PCR分析,筛选获得了同时含有Bar基因和GNA基因的纯合转基因水稻。抗性鉴定表明,褐飞虱在转基因水稻上的进食量显著低于未转化对照和感虫对照品种台农本地1号(TN1),褐飞虱在转基因水稻上的繁殖率亦显著低于未转化对照,即转基因水稻对褐飞虱的进食量和繁殖率有明显的抑制作用;转基因水稻4901-1苗期对褐飞虱的抗性达到了中抗水平,较未转化对照扬辐粳8号的感虫水平有了明显提高。4、聚合多个抗性基因培育多抗转基因水稻。利用双菌双载体介导的共转化,将AP1基因、Bt基因和HPT基因同时导入粳稻品种广陵香粳中,并将其与同时含有GNA和Bar基因的转基因水稻杂交,从其自交后代中筛选获得了同时含有两个、三个或四个目的基因、但无HPT基因的多种类型的多价转基因水稻材料。通过抗性鉴定试验表明,这些多价转基因水稻表现出预期的抗病和/或抗螟虫和/或抗飞虱和除草剂的多抗特性,具体表现为:(1)含AP1基因的多价转基因水稻对白叶枯病(KS-1-20)的抗性级别与只含AP1基因的单价转基因水稻的抗性级别相同,均由未转化对照的中抗上升为抗病水平,但多价转基因水稻的病斑长度较只含AP1基因的单价转基因水稻有极显著地减小,且GNA基因和Bar基因对白叶枯病抗性的提高程度要大于Bt基因;用4个白叶枯病菌种混合后接种,同样表现为GNA基因和Bar基因可提高含AP1基因转基因水稻对白叶枯病的抗性;含AP1基因多价转基因水稻对纹枯病的抗性级别由未转化对照的中感上升为抗病,且GNA和Bar基因对纹枯病的抗性的提高程度大于Bt基因;(2)含Bt基因的多价转基因水稻对稻纵卷叶螟、二化螟抗性较未转化对照有了明显提高,具体表现为,稻纵卷叶螟对含Bt基因的多价转水稻离体叶片的危害程度明显小于未转化对照,含Bt基因的多价转水稻离体叶片上的稻纵卷叶螟幼虫全部死亡;含Bt基因的多价转水稻对二化螟的抗性由未转化对照的高感上升为抗或中抗级别;在不施用任何农药的田间自然条件下,含Bt基因的多价转基因水稻表现为无稻纵卷叶螟危害,未转化对照的受害率达100%;(3)褐飞虱取食含GNA基因的多价转基因水稻后排泄的蜜露量均较未转化对照和感虫对照品种TN1有极显著的减少,含GNA基因的不同多价转基因水稻上褐飞虱排泄的蜜露量差异不显著。5、转反义Wx基因水稻及其品质分析。从通过双菌双载体或超双元载体介导的共转化获得的武香粳9号、协青早和龙特甫转反义Wx基因水稻的自交后代中,筛选获得了无HPT基因的转反义Wx基因水稻。对转反义Wx基因水稻的分析表明:(1)反义Wx基因导入水稻后,可抑制转基因水稻胚乳中内源Wx基因的表达,表现为未成熟种子胚乳中的成熟2.3-kbWx基因RNA以及未剪切的3.3-kbWx基因RNA量均较未转化对照降低,成熟种子胚乳中的Wx蛋白表达量,直链淀粉含量较未转化对照有不同程度的降低,且在粳稻和籼稻中都选育获得了直链淀粉含量降低到糯性水平的转基因水稻植株;(2)直链淀粉含量降低后,对种子的其它淀粉品质性状也有明显的影响,表现为胶稠度变软;糊化温度在不同品种中表现不同,武香粳9号和协青早的转基因水稻,直链淀粉含量下降后,糊化温度变化不显著,龙特甫的转基因水稻,当直链淀粉含量明显下降后,糊化温度极显著升高;转基因水稻中的直链淀粉含量降低后对淀粉粘滞性谱(RVA)特性也有一些影响,但不同来源的转反义Wx基因水稻的表现不完全一致。当直链淀粉含量降低到糯性水平时,转基因水稻RVA谱的PKV、HPV和CPV均极显著降低;(3)当转基因水稻籽粒中直链淀粉含量极显著降低时,淀粉粒的充实情况变差;(4)转基因水稻直链淀粉含量降至糯性水平时,千粒重明显降低,最多可降低17.05%。6、转全长Wx基因水稻及其品质分析。对EHA105/p13W2介导获得的协青早、龙特甫、武香粳9号、武香9915和广陵香糯的转全长Wx基因水稻的分析表明:(1)全长Wx基因导入水稻后,大部分转基因水稻中Wx基因的表达量提高,表现为未成熟种子胚乳中的2.3-kbWx基因RNA量较未转化对照升高,成熟种子胚乳中的Wx蛋白表达量,直链淀粉含量较未转化对照有不同程度的升高,糯稻、粳稻和籼稻的转基因水稻的直链淀粉含量分别可达24.92%、24.14%,和27.52%,分别较对照提高了22.91%、9.72%和2.99%,即与籼稻相比,糯稻和粳稻品种转基因水稻中升高的幅度较大;(2)直链淀粉含量提高后,糯稻和粳稻转全长Wx基因水稻的胶稠度变硬,而籼稻转全长Wx基因水稻因其直链淀粉含量变化较小,胶稠度也无显著变化;广陵香糯来源的转基因水稻的糊化温度极显著降低,而其余四个品种转基因水稻的糊化温度均未发生显著变化;不同品种的转全长Wx基因水稻的RVA谱与未转化对照相比,表现出不同的差异;(4)粳稻和糯稻的转全长Wx基因水稻的直链淀粉含量升高幅度较大时,淀粉粒的充实情况变好,籼稻的转全长Wx基因水稻,因其直链淀粉含量升高幅度较小,其淀粉粒的变化亦较小;(5)糯稻和粳稻的转全长Wx基因水稻,当直链淀粉含量提高明显时,糙米千粒重有所提高,最高可较对照增加16.89%。7、糯稻中表达Wx cDNA及其对稻米品质的影响。以双菌双载体介导的共转化法,将Wx cDNA和HPT基因同时导入到了广陵香糯和苏御糯中。对转Wx-cDNA水稻成熟种子的分析结果表明:(1)大部分转Wx cDNA水稻胚乳中的Wx蛋白表达量提高,直链淀粉含量有不同程度的提高;(2)直链淀粉含量提高后,转Wx cDNA水稻的胶稠度变硬,糊化温度的变化在不同转化子中表现不完全一致,有升高也有降低;(3)当转Wx cDNA水稻的直链含量提高时,其RVA谱的特征值PKV、HPV和CPV均较相应的未转化对照大;(5)当直链淀粉含量明显升高时,转Wx cDNA水稻的淀粉粒的充实情况变好,糙米千粒重有所提高,最高可较对照增加8.56 %。更多还原

【Abstract】 Rice is one of the most important crops in the world, providing staple food for more than half of the world’s population. It have got great achievement for improvement of rice yielding, resistance and quality through conventional breeding approach, but it becomes much difficult to further increase rice yielding and quality as the limition of conventional breeding and genetic resource. Recent advancements in molecular genetics and biotechnology offer new opportunities for rice breeders to increase rice resistance to diseases, insects and herbicide or enhance rice starch quality through transformation.The objective of this research was to study improving rice resistance and grain quality of several elite rice varieties through genetical engineering. To increase the resistance to diseases, insects and herbicide in rice, four target genes were used, which include the Bt cryIA(c) gene from Bacillus thuringiensis, GNA (Galanthus nivalis agglutinin) gene from snowdrop, AP1(Amphipathic protein 1)gene from sweet pepper (Capsicum annuum L.) and the herbicide resistance Bar gene from Streptomyces hygroscopicus. The another work was to regulate Wx gene expression in rice grains to improve starch quality via transformation of different Wx constructs, such as the antisense RNA structure, full length of Wx genome sequences and Wx-cDNA. The main results obtained were showed as follows: 1. Bt gene transgenic rice. To produce selectable marker-free transgenic rice lines with increased resistance to pests, calli derived from immature seeds of two elite japonica rice varieties, Guanglingxiangjing and Wuxiangjing 9, were used for Agrobacterium-mediated co-transformation of the Bt cryIA(c) and hygromycin phosphotransferase (HPT) genes. Two different transformation approaches, a single twin T-DNA binary vector in one agrobacterial strain(EHA105/pSBBt) or two separate binary vectors in two separate agrobacterial culture(sEHA105/p03Bt:EHA105/pCAMBIA1300) were used. The twin T-DNA binary vector was composed of two separate T-DNA regions, one carrying the Bt cryIA(c) while the other the HPT gene. The two separate binary vectors either contained the Bt cryIA(c) gene or the HPT gene in individual plasmid. Many transgenic rice lines co-transformed with both the target and HPT genes were obtained, and, subsequenctly, the selectable marker-free Bt transgenic rice lines were selected from the offspring of the co-transgenic plants. The resistance evaluation indicated that: (1) The affected area of the Bt transgenic rice lines leaves in vitro caused by rice leaf folder was smaller than that of the wild types, and all the larvas were dead after 3 dayes feeded on the Bt transgenic rice lines leaves. Affected area of the Bt transgenic rice lines culms in vitro caused by five instar striped stem borers was also smaller than that of the wild types, and the excretion of striped stem borers was little on the Bt transgenic rice lines culms. (2) All the striped stem borer larvas were dead 3 dayes after feeded on the Bt transgenic rice seedlings. The dead hart rate of the Bt transgenic rice lines caused by striped stem borers during maturing stage was lower than that of the wild types, and the resistance grade of some Bt transgenic rice lines reached to middle resistance or resistance level. In natural condition, no Bt transgenic rice lines was disserved by rice leaf folder, and all the wild types were disserved.2. AP1 gene transgenic rice. To produce selectable marker-free transgenic rice lines with increased resistance to diseases, calli derived from immature seeds of Guanglingxiangjing and Wuxiangjing 9 were used for co-transformation of the 14 AP1 and HPT genes mediated with the twin T-DNA binary vector pSBAP1. Many transgenic rice lines co-transformed with the AP1 and HPT genes were obtained and HPT gene free AP1 transgenic rice lines were selected from the offering of the co-transgenic plants. The resistance evaluation after inoculation indicated that the AP1 gene confered the transgenic rice lines improved resistance both to bacterial leaf blight and sheath blight. (1)The bacterial leaf blight lesion length of AP1 transgenic rice lines was shorter than that of the wild types. And the resistance grade of the transgenic rice lines reached to resistance level; (2) Despite the disease resistance grade to sheath blight of the transgenic lines and the wild type was at the same level, middle sensitive, the ratio of the lesion length divided by plant height was reduced significantly compared to the wild type, i.e. the distance the lesions moved up the stem was decreased in the transgenic plants. The resistance of most AP1 transgenic rice lines to false smut was also improved in natural condition.3. GNA and Bar genes transgenic rice. To produce transgenic rice lines with increased resistance to brown planthopper and herbicide, calli derived from immature seeds of Guanglingxiangjing, Wuxiang9915 and Yangfujing 8 were used for transformation mediated with the binary vector pCUGNA-BAR. The resistance evaluation indicated that the GNA and Bar genes confered the transgenic rice lines improvemed resistance both to BPH and herbicide Basta. Honeydew production of BPH feeding on transgenic lines was signifcant lower than that of the wild types and the sensitive control TN1. The propagating frequecy of BPH feeding on the transgenic lines was also lower than that of the wild type. The resistance of one transgenic line, 4901-1, to BPH at seedling stage reached to middle resistance grade.4. Multi-resistance transgenic rice. To produce marker-free transgenic rice lines with improved resistance to pests, diseases and herbicide, calli derived from immature seeds of Guanglingxiangjing were used for co-transformation of the Bt cryIA(c), AP1 and HPT genes mediated with EHA105/pSBAP1 and EHA105/p03Bt. Some of the transgenic rice lines co-transformed with the Bt cryIA(c), AP1 and HPT genes were obtained. These plants were crossed with transgenic rice lines containing both GNA and Bar genes, and, therefore, the HPT gene free transgenic plants with different combination of target genes were selected from their offspring. The resistance evaluation showed that: (1) The AP1 gene confered the multi-gene transgenic rice lines improved resistance both to bacterial leaf blight and sheath blight. The leasion length of multi-gene transgenic plants caused by bacterial leaf blight(KS-1-20) was shorter than that of only AP1 gene transgenic rice. And the leasion length of AP1 gene, GNA gene and Bar gene transgenic rice lines was shorter than that of AP1 and Bt genes transgenic rice; After inoculated with the mixture of four bacterial leaf blight strains, the resistance to bacterial leaf blight was enhanced in multi-transgenic rice lines containing AP1, Bt, GNA and Bar genes when comparing with that of the transgenic lines containing the AP1 and Bt genes; (2) The sheath blight leasion length of multi-gene transgenic plants was shorter than that of the wild type and only AP1 gene transgenic rice. The resistance grades of multi-gene transgenic plants to sheath blight reached to resistance levels. The resistance to sheath blight was enhanced in multi-transgenic rice lines containing AP1, Bt, GNA and Bar genes when comparing with that of the transgenic lines containing the AP1 and Bt genes; (3) The resistance evaluation indicated that the Bt gene confered the multi-gene transgenic rice lines improved resistance to rice leaf folder and striped stem borer. Affected area of the Bt transgenic rice lines leaves in vitro caused by rice leaf folder was smaller than that of the wild type, and all the larvas were dead after 3 dayes feeded on the multi-gene transgenic rice lines leaves. The resistance grade of multi-gene transgenic rice lines to striped stem borers reached to resistance or middle resistance level. In natural condition, no multi-gene transgenic rice lines was affected by rice leaf folder, and all the wild types were affected. (4) The GNA and Bar genes confered the multi-gene transgenic rice lines improved resistance both to BPH and Basta. Honeydew production of BPH feeding on the multi-gene transgenic lines was signifcant lower than that of the wild type and the sensitive control TN1.5. Antisense Wx transgenic rice lines and their quality performance. In order to obtain marker-free and quality improved rice, HPT and anti-Wx genes were co-transformed to Xieqingzao, Longtefu and Wuxiangjing 9 mediated with EHA105/p13W0 and EHA105/pCAMBIA1300 or EHA105/pYH592. Several transgenic lines without the HPT gene were selected from the offspring of the co-transgenic plants. The analyses of Wx gene expression and starch quality indicated that: (1) The expression of Wx gene was suppressed to different level in different transgenic lines, and the amount of mature 2.3kb Wx mRNA and un-spliced 3.3kb Wx pre-mRNA in developing seeds of transgenic rice lines was lower than that of the wild types. The amount of Wx protein and amylose content in mature seeds of transgenic rice lines was also reduced in different transgenic lines, and some of which was reduced to the level of waxy rice, both in japonica rice and indica rice. (2) The other quality was also changed after the amylose content reduced, such as gel consistency (GC) changed to softer. The gelatination temperature (GT) of the transgenic rice lines which derived from Longtefu was increased as their amylose content reduced to that of the waxy rice. But the variance was not notable between the GT of the transgenic rice lines which derived from Wuxiangjing 9 and Xieqingzao and that of their wild types. There was also some variance in RVA profile in the anti-Wx transgenic rice. The PKV, HPV and CPV of the transgenic rice lines were reduced significantly as their amylose content was reduced to that of waxy rice. (4) The starch granules of the transgenic rice lines were not filled as good as that of the wild type when their amylose content was reduced notably. (5)The grain weight was reduced obviously in the transgenic rice lines as their amylose content was reduced to that of waxy rice and the decrease reached to 17.05% compared to that of the wild type. 6. Sense Wx gene transgenic rice lines and their quality performance. The homozygous Wx gene transgenic rice lines were selected from the offspring of the transgenic plants mediated with EHA105/p13W2 derived from Xieqingzao, Longtefu,Wuxiangjing 9, Wuxiang 9915 and Guanglingxiangnuo. Wx gene expression and starch quality analysis indicated that: (1)The expression of Wx gene, as well as the amylose content, was increased to different level in different transgenic rice. The quantity of mature 2.3kb Wx mRNA in transgenic rice lines immature seeds was enhanced while the amylose content was increased. The Wx protein amount and amylose content in transgenic rice lines mature seeds were also enhanced and the enhancement was more obvious in the transgenic rice lines which derived from japonica rice and waxy rice than that from indica rice. For instance, the highest amylose content of transgenic rice lines derived from waxy rice, Japonica rice and indica rice reached to 24.92%, 24.14% and 27.52%,and the enhancement reached to 22.91%, 9.72% and 2.99%,respectively; (2) The other quality was also changed when the amylose content increased, and there were different exhibition in the different transgenic lines which derived from indica rice or japonica rice, such as gel consistency (GC) changed to harder in the transgenic rice lines which derived from japonica rice and waxy rice, but the variance was not obvious in the transgenic rice lines which derived from indica rice. The GT of the transgenic rice lines only which derived from Guanglingxiangnuo was reduced as their amylose content was increased. There were also some variances in RVA profile in different transgenic lines; (3) The starch granules of the transgenic rice lines were filled better than that of the wild types as their amylose content was increased obviously; (4)The 1000-grain brown rice weight was increased in the transgenic rice lines which derived from japonica rice and waxy rice when their amylose content was increased notably, and the enhancement reached to 16.89%.7. Wx cDNA transgenic rice lines and their quality performance. In order to study the effect of Wx cDNA on rice quality, Wx cDNA and HPT gene were co-transformed to Guanglingxiangnuo and Suyunuo mediated with EHA105/pCAMBIA1300 and EHA105/p585. The analyses of Wx gene expression and starch quality indicated that: (1)The Wx protein quantity in the transgenic rice lines mature seeds was enhanced as their amylose content was increased; (2)The amylose content in most of the transgenic rice lines was increased differently in different transgenic rice lines. The highest one reached to 19.12% and the enhancement reached to 17.82%; (3) The other quality was changed as their amylose content increased. GC was changed to harder in the transgenic rice lines as their amylose content was increased. But the variance of GT was complex, i.e. some of them were increased and some of them were decreased; (4) There were also some variance in RVA profile in different transgenic lines as their amylose content was increased, such as PKV, HPV and CPV of the transgenic rice lines were higher than that of the wild type. (5) The starch granules were filled better when their amylose content was increased obviously in the transgenic rice; (6)The 1000-grain brown rice weight was increased in the transgenic rice lines when their amylose content was increased notably, and the enhancement reached to 8.56 %.更多还原