【作者】 燕树锋；

【导师】 祝水金；

【作者基本信息】 浙江大学 ， 作物学， 2011， 博士

【摘要】 杂草是棉花生产主要障碍之一,棉花杂草为害常年造成损失为14%-16%。人工除草是我国控制草害的传统方法。随着城市化进程的加速,农村人口的转移以及劳动力成本的提高,棉田大面积人工除草已不能适应现代农业生产的需求。草甘膦是一种灭生性除草剂,具有广谱、高效、低毒和无残留的特点,其销售额占整个除草剂市场总量的30%,已连续多年占据世界农药销售额首位。抗草甘膦棉花品种是实现田间化学除草的先决条件,培育具有我国自主知识产权的抗草甘膦棉花品种则对于我国棉花产业具有重要的实践意义。本试验以珂字棉201和中棉所49为受体材料,分别通过农杆菌介导法和花粉管通道法两种方法,转化由本校自主研发并具有自主知识产权的抗草甘膦基因EPSPs-G6,创造抗草甘膦的转基因棉花种质资源,为培育具有我国自主知识产权的抗草甘膦转基因棉花品种提供新的种质。同时,对育成的转基因抗草甘膦棉花种质材料进行综合鉴定和评价,为该种质育种利用和抗草甘膦棉花的商业化生产提供科学依据。主要研究结果如下：1.转基因抗草甘膦棉花种质的创造本研究以珂字棉201下胚轴作为外植体,用50 mg.L-1卡那霉素作为抗性愈伤组织的筛选剂,将含有玉米泛素Ubi启动子驱动的EPSPS-G6基因通过农杆菌介导法导入棉花基因组,获得了2株T0代转基因植株,2株转基因植株开花正常,自交种子种成株行(T1)。经过PCR检测证实EPSPS-G6基因已经导入棉花基因组,但对草甘膦的抗性较差,可能与来源于单子叶玉米的Ubi启动子在棉花中驱动EPSPS-G6基因表达效率有关。通过花粉管通道法将Camv 35S启动子驱动的EPSPS-G6基因导入陆地棉品种中棉所49,共获得26个具有草甘膦抗性的转化植株。PCR检测、Southem点杂交、Southem杂交(双酶切和单酶切)均证实EPSPS-G6基因已经导入棉花基因组。T1田间抗性鉴定结果表明26个转化子中20个转化子经卡方测定符合一对基因的分离规律。经T2田间抗性鉴定,共获得152个纯合材料,分别来源于26个转化子。2.田问抗性鉴定各试验材料分别用15 mM、20 mM、30 mM和40 mM浓度的草甘膦进行涂抹鉴定。非转基因的中棉所49和TM-1对草甘膦十分敏感,15 mM浓度草甘膦处理下死亡。本研究获得的不同转基因材料对草甘膦抗性存在着显著差异,其中采用花粉通管法获得的26个转化子在20 mM田间使用草甘膦浓度下均无反应,但在30 mM草甘膦浓度下,EHC09-045和EHC09-029发生萎蔫或轻萎蔫,但都得到恢复。在40 mM草甘膦浓度下,有11个转化子的幼苗仍无反应,表现为高抗草甘膦；13个转化子发生轻萎蔫或萎蔫,但恢复,表现为中度抗草甘膦；2个转化子死亡,抗草甘膦性较弱。3.抗草甘膦性状的遗传分析选用5个转基因抗草甘膦棉花种质系(EHC09-002、EHC09-009、EHC09-036、EHC09-038和EHC09-045)进行遗传分析,结果表明5个转基因抗草甘膦棉花种质系均不受细胞质效应的影响,EHC09-002,EHC09-009和EHC09-036三个转化子与TM-1和中棉所49正反交F2,经χ2检验符合3：1的分离规律,分子检测表明这三个转化子都是单基因插入的,与遗传研究相符。但EHC09-038和EHC09-045两个转化子经χ2检验不符合3：1的分离规律,这可能与基因的插入位点和基因的整合有关。4.转基因抗草甘膦棉花种质系的农艺性状和纤维品质本研究中通过花粉管通道法获得的26个转化子,农艺性状和纤维品质总体表现较好,部分转化子表现优于对照。T,农艺性状考察结果表明转基因抗草甘膦棉花株高、果枝数和单株铃数三项指标与对照相比差异不大,但外源基因的导入对转基因抗草甘膦棉花的衣分影响较大。T2农艺性状和纤维品质考察结果表明外源基因的导入对纤维长度和整齐度影响不大,而对麦克隆值、伸长率和断裂比强度等纤维品质指标,以及铃重和衣分等农艺性状均有较大影响。因此,转基因棉花育种研究中不仅只注重目标性状的转育,还要注重非目标性状的筛选。5.市售草甘膦对转基因抗草甘膦棉花幼苗生长的影响以转基因抗草甘膦棉花种质系EHC09-008和EHC09-020及其非转基因遗传背景亲本对照中棉所49为材料,研究了市售10%草甘膦水剂和95%的草甘膦粉剂对抗草甘膦棉花种子萌发和幼苗生长的影响。研究结果表明,10%的草甘膦水剂中可能含有其他除草剂或对棉花幼苗生长发育有害的物质,影响抗草甘膦棉种子的萌发和幼苗的生长。因此,在转基因抗草甘膦棉花生产应用时,应必谨慎选用草甘膦除草剂产品。更多还原

【Abstract】 weed infestation constitutes a major hazard in cotton production resulting in about 14-16% loss in yield. Manual tending is a primitive and traditional method for weeds control. However, due to the accelerated urbanization process, the transfer of rural population and the increase in labor cost, manual tending can no longer meet the needs of modern agricultural production. Glyphosate is a non-selective herbicide. Because of its broad-spectrum effect, high efficiency, and low toxicity and residual effect, it occupies 30% of the total herbicide market and has ranked the first agrochemical for many years. Selection and breeding glyphosate-resistant cotton varieties is the prerequisite to achieve chemical weed controlling on the cotton field. It is very necessary to breed our own patent of glyphosate-tolerant GM cotton for cotton production in China. In this study, Glyphosate-resistant EPSPS-G6 gene, which was cloned by Zhejiang University, was introduced into upland cottons culitivars, using coker 201 and CCRI-49 as the receptors by Agrobacterium- mediated method and pollen tube pathway method, and glyphosate-resistant transgenic cotton germplasms were obtained, which provided the new germplasm for development of glyphosate-tolerant cotton cultivars with our own intellectual property rights. Meanwhile, comprehensive identification and evaluation of germplasms provided a scientific basis for breeding and commercial production of transgenic glyphosate-resistant cotton. The major results in this study are as follows: 1. Developing of transgenic glyphosate-resistant cotton germplasmsThe EPSPS-G6 gene driven by ubiquitin promoter from maize was introduced into the upland cotton through agrobacterium- mediated method using coker 201 hypocotyls as explants and 50 mg.L-1 kanamycin as screening agent. Two T0 transgenic plants were obtained and the T1 lines was planted from the self-crossed seeds. The EPSPS-G6 gene was confirmed by PCR in the T0 plants and their T1 lines. But the resistance to glyphosate of the transgenic plants by agrobacterium-mediated method was not satisfied, probably because the Ubi promoter derived from monocot maize was not effective enough in expression of EPSPS-G6 gene in dicot cotton.Another vector with the EPSPS-G6 gene driven by Camv 35S promoter was introduced into CCRI-49 by the pollen tube pathway, and 26 transformants with glyphosate-resistance were obtained. PCR, dot blot hybridization, Southern hybridization (single and double restriction enzymes digestion) showed successful integration of the foreign EPSPS-G6 gene into the cotton genome. Field resistance test in T1 plants showed that the 20 of 26 transformants were consistent with single-gene Mendelian inheritance.152 homozygous resistance lines were obtained derived from 26 transformants, respectively. 2. Field test of glyphosate-resistanceThe test materials were treated with 15 mM,20 mM,30 mM and 40 mM of glyphosate. Non-transgenic CCRI-49 and TM-1 is highly sensitive to glyphosate herbicide, and their plants were died even treated by 15 mM glyphosate. While the transgenic plants obtained from the pollen tube pathway expressed glyphosate resistance with different levels. All the transgenic cotton plant were not affected with the treatment of 20 mM glyphosate, while a light wilting occurred in two transformants (EHC09-045 and EHC09-029) when they were treated with 30 mM glyphosate. At the treatment of 40 mM glyphosate, the resistance to glyphosate were greatly difference among the transformants,11 transformants of them showed no reaction at all, which indicated to be high resistant ones.13 transformants initially wilting or light wilting but recovered eventually, which shown moderate resistant ones; and 2 transformants died which indicated to be low resistant.3. Genetic analysis of glyphosate-resistant trait in transgenic cottonReciprocal crosses of five selected transgenic cotton lines, namely EHC09-002, E HC09-009, EHC09-036, EHC09-038 and EHC09-045, with TM-1 and CCRI-49 were performed. The glyphosate resistant trait of the transgenic cotton in F1 showed that there was no cytoplasmic effect as the same results of the crosses used the transgenic cotton germplasm either as female parent or male parent. The separation and distribution of the glyphosate resistant trait in F2 showed that EHC09-002, EHC09-009 and EHC09-036 were consistent with 3:1 ratio, according toχ2 test and confirmed by southern blot. However, EHC09-038 and EHC09-045 were inconsistent with the ratio 3:1, probably due to gene insertion sites and the integration of the gene.4. Agronomic traits and fiber quality in transgenic glyphosate-resistant cottonThe overall performance of agronomic traits and fiber quality of 26 transformants obtained by pollen tube pathway was normal and some even better than the control. The agronomic traits of T1 showed that plant height, fruiting branch number and boll number were not significantly different from those of the control. However, the integration of exogenous gene into cotton had a great impact on lint percentage and boll weight. The results in T2 plants showed that the transgenic cotton had little impact on fiber uniformity and fiber length, but had major impact on micronaire value, fiber elongation, and fiber strength, It suggested that the breeding of the transgenic cotton should focus on not only goal-oriented characters but also non-target traits during the selection.5. The effects of commercial glyphosate on the growth of the transgenic cotton seedlingsThe effects of three commercial glyphosates, (two 10% glyphosate aqueous solution and one 95% glyphosate powder) on the seed germination and seedling growth of transgenic cotton with glyphosate resistance were studies, using the transgenic glyphosate-resistant cotton, EHC09-008 and EHC09-020, as the materials, and their non-transgenic genetic background cultivar CCRI-49 as control. The results showed that 10% glyphosate aqueous solution may contain some harmful substances and affect the growth and development of the transgenic glyphosate-resistant cotton seedlings. Therefore the kinds of glyphosate should be selected carefully when they were used in chemical weed controlling in the transgenic glyphosate-resistant cotton field.更多还原