首页>>学术检索

大豆子叶节转化体系的优化及抗逆基因AtNHX5的遗传转化研究

标题: 大豆子叶节转化体系的优化及抗逆基因AtNHX5的遗传转化研究
英文标题: The Research on Optimization of Soybean Cotyledonary Node Regeneration and Genetic Transformation of Resistance Gene AtNHX5
作者: 杨权
出版时间: 2012-01-01
所在大学: 河北农业大学
关键词: 大豆,子叶节,AtNHX5基因,遗传转化,除草剂
英文关键词: soybean,cotyledonary node,AtNHX5,genetic transformation,phytocide
论文级别: 硕士
学位: 学位论文
导师: 王冬梅
专业: 植物学
提交时间: 2012
摘要: 大豆作为世界上重要的粮食和油料作物,其产量和品质由于环境的影响均受到一定制约.在我国土壤盐碱化是限制大豆产量的重要因素之一,盐碱化不仅降低耕地的生产力,而且严重制约着耕地的可持续利用,直接影响农业的可持续发展.因此,抗盐新品种的培育成为近些年来研究的热点和重点.利用农杆菌介导法将抗盐基因转入大豆,综合运用分子生物学方法与转基因技术培育抗盐大豆新品种,将为改良盐碱地提供一条兼有经济、生态效益的有效途径.本研究以河北省推广大豆品种子叶节为受体材料,利用农杆菌介导法进行遗传转化条件的摸索,在此基础上利用优化的子叶节转化体系,进行了抗逆基因AtNHX5(CPA1家族的拟南芥Na+/H+逆向转运蛋白基因)的大豆遗传转化.获得主要结果如下:1、利用3个河北省推广的大豆品种进行了农杆菌介导的大豆子叶节遗传转化条件的摸索,探讨了不同萌发培养基、子叶节苗龄、外植体预处理温度、超声波处理时间及抗氧化剂浓度对农杆菌侵染后子叶节不定芽诱导的影响.结果表明以萌发6 d、4℃低温预处理24 h的大豆子叶节为外植体,农杆菌侵染后经超声波处理30 S、共培养培养基中添加20 mg/L抗氧化剂硝酸银,能明显提高子叶节丛生芽诱导率.对五星2号T0代进行了报告基因GUS的检测,获得PCR验证阳性植株18株,转化率达0.97%.2、以优化的大豆子叶节遗传转化体系,利用农杆菌介导法进行了拟南芥液泡膜钠氢逆向转运蛋白基因AtNHX5的遗传转化.T0代经PCR检测,转化率为0.23%,并通过RT-PCR检测证明目的基因已在转录水平进行了表达.3、对PCR检测为阳性的五星2号T1代幼苗以及对照植株分别滴注100 mg/L Basta进行筛选,发现阳性植株可以将Basta限制在一个较小的范围内,褐斑不扩散集中于注射部位,而对照植株叶片大面积褪绿,甚至整片叶枯黄.进一步证明转AtNHX5基因的五星2号转化植株具有一定的除草剂抗性.4、经Basta筛选及PCR检测均为阳性的五星2号T1代幼苗,经200 mmol/L NaCl胁迫处理后,发现阳性植株仍然能够正常生长,而对照植株叶片已经枯黄.初步证明转AtNHX5基因的五星2号植株具有一定的抗盐性.
英文摘要: Soybean is one of the most important foods and oil crop in the world, but its production and quality is restricted by environment partly. Salinity is a major constraint of crop productivity because it reduces yield and limits expansion of agriculture onto previously uncultivated land in china. For this reason, the incubation of new antireversional variety is becoming an emphasis and a hot research as well for the past few years. So cultivating new antireversional variety of soybean by the Agrobacterium-mediated method is an efficient way to protect the environment and increase economic.In this study, three high-quality soybean varieties in Hebei Province were used as speciments. An efficient Agrobacterium-mediated gene transformation system based on the examinations of several factors affecting plant transformation efficiency was developed. Using the optimized system for the transformation, the wuxing2 cotyledonary node regeneration system as the receptor system, genetic transformation of the Arabidopsis thaliana salt-tolerant gene-AtNHX5(Arabidopsis Na+/H+ antiporter gene in Arabidopsis thaliana CPA1 family)is undertaken using agrobacterium-mediated method. Results are as follows:1. In this study, Soybean cotyledonary node of Hebei excellent cultivars were used as speciments. An efficient Agrobacterium-mediated gene transformation system based on the examinations of several factors affecting plant transformation efficiency was developed for soybean. The results showed that increased transgenic shoots were obtained when seeds germinated 6 days and low temperature pretreatment. The application of 20 mg/L anti-oxidant silver nitrate during co-cultivation can ease browning phenomenon. The optimized ultrasonic treatment for infected explants was 30 S. Using the system for the transformation of wuxing2, the explants were incubated in the buffer for histochemical assay of GUS activity. Finally, we obtained 18 transgenic plants which PCR-positive rate was 0.97%.2. In this study, used the optimized system, investigated AtNHX5 transformation by cotyledonary node method. The result showed that the AtNHX5 had already integrated to the genome of Wuxing2 soybean regeneration plants initially by PCR verification, and the T0 transformation efficiency was 0.23%. The expression of AtNHX5 was assessed by RT-PCR analysis.3. The progeny of control plants and T0 plants which PCR and RT-PCR analysis positive grown in the greenhouse were examined for herbicide resistance. Under Basta concentration of 100 mg/L, we found the positive transgenic plants can limit Basta within a small range, but the control became a large area of death, or even the whole leaf brown. Further evidence showed inheritance in T1 generation plants.4. The progeny of control plants and T0 plants which PCR and RT-PCR analysis positive grown 30 d in the greenhouse were examined for salt stress. Under NaCl concentration of 200 mmol/L for 10 d, we found the positive transgenic plant grew normally, but the control were significantly inhibited. Demonstrated salt-tolerance in T1 generation plants initially.