转PeDREB2a、HhERF2和CP4EPSPS基因棉花新材料的获得与抗性分析
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摘要
棉花属于双子叶植物纲(Dicotylwdoneae),锦葵目(Malvaceae),棉属(Gossypium L.)植物,属世界性的经济作物。近年来,受气候变化的影响,全球水资源短缺、土壤盐渍化导致可利用耕地面积逐年减少,有限的可耕地面积与棉花供求之间的矛盾日益突出。因此,培育抗逆转基因棉花新品种,提高棉花抗胁迫能力的需要越来越迫切。随着生物技术的快速发展,从分子水平上研究棉花与非生物逆境之间的关系,利用基因工程技术创制棉花新材料,有望大大加速棉花抗逆新品种的培育进程。本试验通过将来自于新疆戈壁沙生植物胡杨(Populus euphratica)和铃铛刺(Halimodendron halodendron (Pall.)Voss.)的耐旱耐盐转录因子基因PeDREB2a、HhERF2及抗除草剂基因CP4EPSPS转化陆地棉栽培品种WJ、WSH、WY,以期提高棉花耐旱耐盐抗除草剂的能力。主要研究结果如下：
(1)构建了含有PeDREB2a、HhERF2、CP4EPSPS的三价基因表达载体pCAMBIA2301-Kanamycin-CP4EPSPS-HhERF2:PeDREB2a。利用非组培农杆菌介导法将表达载体转化棉花品种WJ、WSH和WY。
(2)建立了棉花室内生长系统。室内每株转基因棉花平均结桃数可达8-10个,每株棉花平均结籽50粒左右,解决了室内棉花发芽率低的问题。
(3)经过大田筛选及分子检测,获得7株T1代转基因植株。经过严格自交及筛选、淘汰,获得T2代转基因株系2个,分别命名为WJ17-24和WJ17-25-2。并于2012年4月向农业部提交了《转HhERF2、PeDREB2a和CP4EPSPS基因抗逆耐除草剂棉花WJS-1等在北京市的中间试验安全评价报告书》,获农业部批准(农基安办字2012-T159)。
(4)对所获得的转基因棉花新株系进行田间农艺性状的观测,发现转基因植株较对照具有显著的矮化及高产性状：平均株高由122.13厘米矮化至88.3厘米；节间长由10.74厘米缩短至8.61厘米；铃数由19个增加至25个。
(5)对转基因植株进行干旱、盐胁迫发现,转基因植株的耐旱性有所提高；对干旱、盐胁迫下对叶片相对含水量、组织丙二醛含量、可溶性糖含量等生理指标综合分析发现,转基因植株耐盐性得到增强；对RNA表达上平上进行分析发现,干旱、盐诱导下PeDREB2a、HhERF2转录因子基因在不同时期内根和叶中均有表达。
(6)通过除草剂试验,确立了0.4%的草甘膦浓度为大田棉花的抗性筛选浓度；0.8%的草甘膦浓度为对照植株的致死浓度；确定了1.0%的草甘膦浓度为转基因植株的致死浓度。
Cotton is a crop plant belongs to the mallow family (Malvaceae) Gossypium of (Gossypium). Cotton is a colossal special commodity related to the agriculture and textile industry, also is original material for defense, medicine, and other aspects of the automotive industry. With the increasingly serious environmental problems in human society, global cotton production is seriously decreased. The main results are listed as follows:
(1) The target gene HhERF2and PeDREB2a were inserted into the pCAMBLA2301-muti cloning site, and other elements such as omega、Poly4and NOS were also included in this vector, thus a trivalent vector pCAMBLA2301-Kanamycin-CP4-HhERF2:PeDREB2a was obtained. The Trivalent vector was transformed into Agrobacterium tumefaciens LBA4404using the heat shock method. The trivalent expression vector was transformed into cotton varieties WJ. WSH and WY using the non-tissue culture Agrobacterium-mediated method.
(2) In order to effectively transform and screen the transgenic cotton, green house growing system was established, the conditions of the cultural temperature, humidity, light, cotton potted soil ratio were characterized. The herbicide resistance experiment was also performed in green house, by spraying different concentrations of glyphosate to cotton. The wilting index was determined based on the damage symptoms.
(3) The TO generation seeds were sown in field. The seeds were harvested from7Tl transgenic plants which confirmed by PCR and glyphosate resistance assay. Tl transgenic plants were self-pollinated strictly, and finally two genetically modified cotton plants were obtained, named WJ17-24and WJ17-25-2.respectively.
(4) Observations of the agronomic traits of WJ17-24-2and WJ17-25-2. the results showed that the plant height of transgenic plant was significantly lower than the control plant. And the stem was also shorter as compared to the control stem was also shorter compared the wild type.
(5) The transgenic plants performed higher salt-tolerant As compared to the control. The gene expression levels of PeDREB2a and HhERF2were higher under the salt and drought stress condition Comparing to to the control, and the expression levels in flowering and boll stage were higher than in the seedling and boll opening stage.
(6) Transgenic and control plants sprayed with different concentrations of herbicides found that the transgenic plants compared with the control has significant herbicide-resistant traits0.4%glyphosate concentration was confirmed to screen the transgenic cotton through statistic analysis by SPSS16.0software one-way ANOVA..Identified by the lethal concentration of1.0%glyphosate concentration of the transgenic plants, glyphosate concentration of0.8%lethal concentration control plants.
(1)构建了含有PeDREB2a、HhERF2、CP4EPSPS的三价基因表达载体pCAMBIA2301-Kanamycin-CP4EPSPS-HhERF2:PeDREB2a。利用非组培农杆菌介导法将表达载体转化棉花品种WJ、WSH和WY。
(2)建立了棉花室内生长系统。室内每株转基因棉花平均结桃数可达8-10个,每株棉花平均结籽50粒左右,解决了室内棉花发芽率低的问题。
(3)经过大田筛选及分子检测,获得7株T1代转基因植株。经过严格自交及筛选、淘汰,获得T2代转基因株系2个,分别命名为WJ17-24和WJ17-25-2。并于2012年4月向农业部提交了《转HhERF2、PeDREB2a和CP4EPSPS基因抗逆耐除草剂棉花WJS-1等在北京市的中间试验安全评价报告书》,获农业部批准(农基安办字2012-T159)。
(4)对所获得的转基因棉花新株系进行田间农艺性状的观测,发现转基因植株较对照具有显著的矮化及高产性状：平均株高由122.13厘米矮化至88.3厘米；节间长由10.74厘米缩短至8.61厘米；铃数由19个增加至25个。
(5)对转基因植株进行干旱、盐胁迫发现,转基因植株的耐旱性有所提高；对干旱、盐胁迫下对叶片相对含水量、组织丙二醛含量、可溶性糖含量等生理指标综合分析发现,转基因植株耐盐性得到增强；对RNA表达上平上进行分析发现,干旱、盐诱导下PeDREB2a、HhERF2转录因子基因在不同时期内根和叶中均有表达。
(6)通过除草剂试验,确立了0.4%的草甘膦浓度为大田棉花的抗性筛选浓度；0.8%的草甘膦浓度为对照植株的致死浓度；确定了1.0%的草甘膦浓度为转基因植株的致死浓度。
Cotton is a crop plant belongs to the mallow family (Malvaceae) Gossypium of (Gossypium). Cotton is a colossal special commodity related to the agriculture and textile industry, also is original material for defense, medicine, and other aspects of the automotive industry. With the increasingly serious environmental problems in human society, global cotton production is seriously decreased. The main results are listed as follows:
(1) The target gene HhERF2and PeDREB2a were inserted into the pCAMBLA2301-muti cloning site, and other elements such as omega、Poly4and NOS were also included in this vector, thus a trivalent vector pCAMBLA2301-Kanamycin-CP4-HhERF2:PeDREB2a was obtained. The Trivalent vector was transformed into Agrobacterium tumefaciens LBA4404using the heat shock method. The trivalent expression vector was transformed into cotton varieties WJ. WSH and WY using the non-tissue culture Agrobacterium-mediated method.
(2) In order to effectively transform and screen the transgenic cotton, green house growing system was established, the conditions of the cultural temperature, humidity, light, cotton potted soil ratio were characterized. The herbicide resistance experiment was also performed in green house, by spraying different concentrations of glyphosate to cotton. The wilting index was determined based on the damage symptoms.
(3) The TO generation seeds were sown in field. The seeds were harvested from7Tl transgenic plants which confirmed by PCR and glyphosate resistance assay. Tl transgenic plants were self-pollinated strictly, and finally two genetically modified cotton plants were obtained, named WJ17-24and WJ17-25-2.respectively.
(4) Observations of the agronomic traits of WJ17-24-2and WJ17-25-2. the results showed that the plant height of transgenic plant was significantly lower than the control plant. And the stem was also shorter as compared to the control stem was also shorter compared the wild type.
(5) The transgenic plants performed higher salt-tolerant As compared to the control. The gene expression levels of PeDREB2a and HhERF2were higher under the salt and drought stress condition Comparing to to the control, and the expression levels in flowering and boll stage were higher than in the seedling and boll opening stage.
(6) Transgenic and control plants sprayed with different concentrations of herbicides found that the transgenic plants compared with the control has significant herbicide-resistant traits0.4%glyphosate concentration was confirmed to screen the transgenic cotton through statistic analysis by SPSS16.0software one-way ANOVA..Identified by the lethal concentration of1.0%glyphosate concentration of the transgenic plants, glyphosate concentration of0.8%lethal concentration control plants.
引文
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