【作者】 蒋玉蓉；

【导师】 祝水金；

【作者基本信息】 浙江大学 ， 作物学， 2007， 博士

【摘要】 棉花是一种重要的经济作物，基因工程技术对于提高棉花的产量、增强抗逆能力和改良纤维品质具有重要的作用。目前用于棉花的转基因系统主要有载体转化系统、DNA直接导入系统和种质转化系统等。种质转化系统（germ line transformation）是利用植物自身的生殖过程转化外源基因，无需经过组培而直接得到转化种子。本文结合农杆菌介导法和花粉管通道法的优点，创建了一种花粉转介外源基因转化的新方法—花粉粒农杆菌转染法，并与棉花的其它转化方法—子房注射质粒DNA、农杆菌介导柱头点滴法和顶端分生组织农杆菌介导法等进行了分析比较，对四种方法的转化效率和转基因植株的遗传特性进行研究；同时，利用获得的转bar基因抗除草剂棉花种质系为亲本进行杂种优势利用及杂种纯度控制的研究，创建出一套可控制田间纯度的杂交棉制种新技术。此外，本研究还通过花粉管通道法将山菠菜BADH基因和拟南芥的AtNHX1基因导入棉花，创造耐盐能力得到较大提高的转基因耐盐棉花种质材料。主要研究结果如下：1．棉花花粉粒农杆菌转染技术的研制花粉农杆菌感染法是通过农杆菌和花粉共培养，再利用自身的花粉管授粉途径转化外源基因的一种新的棉花遗传转化方法。将携带有目的基因质粒pCB4的农杆菌菌株LBA4404与陆地棉新鲜花粉粒共培养于花粉萌发培养基中进行转染，质粒载体pCB4中包括标记基因bar，CaMV 35S启动子及NOS终止子。通过除草剂Basta抗性鉴定，PCR及Southern杂交检测，验证了bar基因的整合和表达。并对转基因棉种质的外源基因遗传特点和对农艺和经济性状的影响进行了研究。结果表明，该方法的转化率达5.5％，显著高于花粉管通道法。分子检测和遗传分析结果表明，外源基因在转基因植株中均为单拷贝，符合典型的孟德尔分离规律，外源基因的整合对于转基因棉的农艺性状和经济性状无显著的影响，是一种具有较好利用价值的棉花外源基因转化方法。2．棉花外源基因转化方法的比较采用包括本研究建立的棉花农杆菌花粉粒介导技术在内的多种外源基因转化方法，用携带有基因bar的质粒pCB4的农杆菌菌株LBA4404转化陆地棉“Coker312”，评价各方法的转化效果和外源基因的遗传特点。结果表明，子房注射质粒DNA（方法Ⅰ，普通的花粉管通道法）、花粉粒农杆菌转染法（方法Ⅱ）、农杆菌介导柱头点滴法（方法Ⅲ）和顶端分生组织农杆菌介导法（方法Ⅳ）均获得了具有外源基因表达的转化植株。叶片除草剂Basta抗性鉴定、PCR检测和Southern杂交证实了bar基因已经整合到植物基因组中。然而，各种方法的转化率、所需要的转化时间以及方法的可重复性和操作简易性各有差异。同其他三种方法相比，方法Ⅱ是一种比较经济和技术可行的转化方法。它所需要的转化时间与方法Ⅰ大致相同，而可重复性接近于方法Ⅳ。另外，方法Ⅱ操作简单经济，无需昂贵的仪器，无需经过方法Ⅳ所要的组织培养。研究结果还表明，虽然方法Ⅳ转化效率比较高，但得到的转基因植株却低于方法Ⅱ，且转基因植株的生长发育受到一定的影响。从各种参考表现来看，方法Ⅲ是所有转化方法中最不可取的方法。3．转BADH和AtNHX1耐盐转基因棉花研究通过花粉管通道法将山菠菜BADH基因和拟南芥的AtNHX1基因导入陆地棉品种中棉所35。PCR检测和Southern杂交证明，BADH和AtNHX1基因已经整合到转基因棉花的基因组中，共获得了5株转BADH基因和9株转AtNHX1基因的转化棉株。盐池抗性鉴定结果表明，转BADH基因棉和转AtNHX1基因棉在含有0.8％NaCl的人工盐池中的发芽率分别为63.4％和82.4％。显著高于未转外源基因的亲本对照（2.4％）。尽管本研究未能对转基因植株中后期的耐盐性进行深入的鉴定和研究，但初步结果表明，转基因植株具有较好的苗期耐盐特性，具有重要的利用价值。4．抗除草剂转基因亲本杂种优势和杂种纯度利用以本研究获得的转bar基因抗除草剂棉花种质系BR001为父本，与不同来源的转基因抗虫棉进行杂交，获得7个转基因抗虫、抗除草剂杂交棉组合。以推广的抗虫杂交棉中棉所29为对照，研究抗虫抗除草剂杂交棉的产量、纤维品质、农艺性状和杂种优势表现，结果表明，7个组合的皮棉产量均表现为显著的超高亲优势，其中6个组合的皮棉产量表现为正向的竞争优贽，且纤维品质和农艺性状优良，加上其带有抗除草剂特性，可进行田间化学除草，显著降低植棉成本。因此，抗虫、抗除草剂杂交棉具有较好的生产应用前景。由于杂交棉父本的抗除草剂性状是一显性性状，F₁仍具有抗除草剂特性，对杂种F₁棉苗喷施除草剂，在防治田间杂草的同时，可自动清除混杂其中的假杂种，确保杂交棉的纯度。本研究用标记性状（色素腺体）对该技术体系进行验证。结果表明，该杂交棉纯度控制技术具有准确、快速、经济、高效等特点，对于杂交棉生产具有重要的利用价值。此外，以本研究建立的杂交棉纯度控制技术为基础，本研究较客观地评价了杂交棉市场的种子纯度现状，并研究了不同杂交棉纯度对于产量和纤维品质的影响。结果表明，当杂交棉纯度低于90％时，其产量和纤维品质显著降低，提高种子的纯度可显著的提高杂交棉的产量和纤维品质。这一结果说明本研究建立的杂交棉纯度控制技术对于提高杂交棉产量和改善杂交棉纤维品质的意义。更多还原

【Abstract】 Cotton, Gossypium spp., is an economically important crop. Genetic engineering plays a major role in further improving the yield, stress-tolerance and quality of fiber. There are mainly three genetic transformation methods to incorporate valuable alien genes into the cotton genome, namely, vector mediated transformation, DNA direct transformation and germ line transformation. The germ line transformation methods avoid tissue culture steps and direct transformation of genes into in-vivo plants. In the research, we took the advantages of Agrobacterium-mediated transformation and pollen tube pathway to develop a new method of direct transformation mediated via the pollen tube pathway. At the same time, the four different transformation methods, that is, ovarian injection of plasmid DNA, pollination by using pollen co-cultivated with Agrobacterium, Agrobacterium drop/dip and apical meristem explants methods were standardized and compared. Transformation efficiency of the four methods and genetic characteristics of transformed plants were evaluated. In addition, homozygous transgenic bar cotton plants were obtained and used as paternal parent to have hybrid production. Heterosis utilization and hybrid purity controlling in virtue of the anti-herbicide transgenic parent plant were simultaneously studied. Thus a new seed production technique was set up to control the purity of hybrid cotton in field. Furthermore, the transformation of BADH and AtNHX1 into cotton by the method of pollen tube pathway was conducted to enhance the salt tolerance in transgenic cotton. The main experimental results are presented as follows:1. Development of Agrobacterium transfected pollen via pollen tube pathway method for genetic transformation of Gossypium hirsutum L.A new method for the genetic transformation of cotton pollen by means of Agrobacterium co-cultivation and pollen tube pathway mediated transformation was reported. The bar gene, which is involved in herbicide tolerance, was transferred into pollen grains of upland cotton. Transformation was carried out in cotton pollen-germinating medium, and transformation was mediated by Agrobacterium strain LBA4404 harboring a vector pCB4, which contains the gene of interest of herbicide resistance, bar gene, constructed with the CaMV 35S promoter and NOS （nopaline synthase） terminator sequences. The integration and expression of bar in the genome of transgenic plants were analyzed with basta bioassay tests, PCR and Southern blot hybridization. At the same time, the heredity, agronomic characters and economic traits of transgenic plants were analyzed and studied. The results indicated that the transformation efficiency was up to 5.5%, which was obviously higher than that of pollen tube pathway. The exogenous gene was confirmed to integrate into the cotton with single-copy. Progeny analysis and molecule testing showed a classical Mendelian pattern of inheritance in transgenic cottons. The integration of exogenous DNA did not show adverse effect on agronomic characters and economic traits of transgenic plants. The method of pollen tube pathway via ovarian injection proved to be a valuable transformation method.2. Comparison of transformation methods for genetic transformation in Gossypium hirsutum L.Protocols for consistent production of transgenic herbicide tolerant upland cotton were established and compared the different transformation methods. Agrobacterium strain LBA4404 harboring a vector pCB4 carrying the bar gene was utilized to transform into upland cotton cultivar ’Coker312’. The transformation efficiency and genetic characters of different transformation methods were evaluated and studied. We obtained transgenic plants in all four methods, that is, ovarian injection of plasmid DNA （method-I）, pollination by using pollen co-cultivated with Agrobacterium （method-11）, Agrobacterium drop/dip （method-Ill） and apical meristem explants （method-IV） method. The herbicide basta leaf sensitivity test, polymerase chain reaction （PCR）, and southern hybridization confirmed that the bar gene was integrated with the plant genome. But they were differed with respect to transformation efficiency, time consume, reproducibility and simplicity of methods. The method-II found to be economically and technically viable compared with other three methods. The time consume by this method-II was on par with method-1 and reproducibility was on par with method-IV. In addition, method-11 used minimum facilities than tissue culture base method-IV. The transformation efficiency was higher in method-IV, but the percentage established transgenic plants were less than that of method-II. Furthermore, the growth and development of transgenic plants were influenced by the transformation of method-lV. Method-Ill was the least choice-able method for cotton transformation due to the inferior performance.3. Study on transformation of salt resistant genes, BADH and AtNHX1, in cottonThe transformation of BADH gene from Atrplex hortensis and AtNHX1 gene from Arabidopsis thalians into upland cotton cultivar-ZMS35 by the method of pollen tube pathway was conducted to enhance the salt tolerance in transgenic cotton. The results of PCR and Southern-blot indicated that the target genes BADH and AtNHX1 had been integrated into the cotton genome. Five transgenic BADH plants and nine transgenic AtNHX1 plants were ultimately obtained. The identification on salt-resistance of T₂ transgenic seeds in artificial experiment plot with 0.8% NaCl suggested that the germination of transgenic BADH and AtNHX1 cotton was 63.4% and 82.4% respectively, which was significantly higher than that of non-transgenic control cottons. However, the identification of salt tolerance during the middle and later stages of transgenic cotton growth period was not done for further investigation because of lack of time. Preliminary results showed that transgenic plants exhibited good salt tolerance during seedling stage and possessed high utilization value.4. Heterosis utilization and hybrid purity controlling in virtue of the herbicideresistant transgenic parent plantThe herbicide resistant transgenic bar gene cotton germplasm, BR001, which was developed in the research, was used as paternal parent to cross with different transgenic Bt insect resistant cultivars. 7 hybrid combinations with pest and herbicide resistant characteristics were obtained. Agronomic characters, fiber qualities and yield traits of 7 hybrid combinations were investigated to analyze heterosis of transgenic cotton, while using the extending transgenic Bt hybrid cotton cultivar, ZMS29, as control （CK）. The results suggested that all hybrid combinations showed significant higher-parental heterosis and 6 combinations showed positive competitive heterosis in lint yield. Chemical herbicides thus could be applied in the farm and to reduce the cotton plant cost, owing to the hybrid combinations carrying anti-herbicide characteristics. Clearly, the inscet and herbicide resistant transgenic cotton with good fiber quality and agronomic traits possessed fine prospects for productive application. Hybrid F₁ displayed herbicide resistant characters because the herbicide resistant character of paternal parent was controlled by one dominant gene. When F₁ hybrid seedlings were sprayed with herbicide to prevent and control the field weeds, false hybrids could be killed and excluded from population hybrid seeds at the same time to guarantee the purity of hybrid cotton. The study demonstrated the practicability of the purity test techniques by utilizing marker characters （pigment gland）. The results indicated that the purity control techniques possessed the advantageous of accuracy, rapidity, economic and high-efficiency. Undoubtedly, it exhibited important utilization value to hybrid cotton production. In addition, the research made an objective evaluation on current market situation of seed purity of hybrid cotton. And the influence on yield and fiber quality by different purity of hybrid cotton was also studied. The results suggested that yield and fiber quality was evidently decreased when the purity of hybrid cotton below 90%. Therefore, purity of hybrid seed improvement could significantly increased yield and fiber quality. The results indicated the significant importance of the purity control techniques established in the study on yield increase and fiber quality improvement of hybrid cotton.更多还原