超表达BOR1基因提高油菜硼效率的研究

Study on Enhancing Boron Efficiency in Brassica Napus Via Overexpression of BOR1

作者: 专业:植物营养学 导师:徐芳森 年度:2010 学位:硕士  院校: 华中农业大学

Keywords

Boron, Brassica napus, BOR1, Transgene, Boron efficiency

        硼是植物生长发育所必需的微量营养元素。硼在土壤中以无电荷形式H3BO3被植物根系吸收利用。在我国,尤其是在长江中下游地区土壤供硼不足现象较为严重,施用硼肥已成为该地区硼敏感作物生产的重要农艺措施,比如油菜和棉花。在拟南芥中研究发现,超表达硼酸转运子基因BOR1能够在缺硼条件下提高植株对硼的吸收转运和种子产量。本研究通过转基因的方法将BOR1导入甘蓝型油菜"Westar10",获得超表达BOR1基因的植株,然后设置不同的表型鉴定实验来研究其在缺硼条件下的长势及对硼的吸收利用和硼效率。获得的主要研究结果如下:1.转基因植株的获得和筛选。通过农杆菌介导的遗传转化法,获得了13个超表达BOR1的转化子。自交繁种后,利用T2代L1,L2,L3三个株系的种子,进行Kan筛选和PCR验证,从中分别获得3、3和5个无分离的家系,并利用其T3代种子进行筛选验证。目前已经获得T4代转基因植株。2.外源基因BOR1的表达及拷贝数鉴定。利用RT-PCR分析转基因植株不同时期外源基因BOR1在RNA水平上的表达,结果表明转基因植株能够稳定表达外源基因;通过Southern杂交分析外源基因的拷贝数,结果显示L1和L2为双拷贝株系,L3为单拷贝株系。3.转基因植株苗期表型鉴定。水培试验结果表明:在严重缺硼水平下(0.15μM),转基因植株的长势比野生型有优势,3个转基因材料的地上部硼含量和累积量与对照野生型相比,均有不同程度的提高,其中L1和L2的地上部硼含量达到野生型的2倍之多,L1、L2和L3的地上部硼累积量均达到野生型的2-3倍。4.T1代和T3代转基因植株全生育期表型鉴定。土培试验结果如下:T1代实验表明,在中度缺硼(0.25 mg/kg)时,L1和L2的单株角果数、单株产量和含油量相对于野生型都明显增加,其中L1单株产量比野生型提高了43%;L3在严重缺硼(0.09 mg/kg)时苗期地上部含硼量和中度缺硼(0.25 mg/kg)时籽粒硼含量显著提高,但单株产量与野生型差异不明显。T3代实验表明,在不同缺硼条件下,L1和L2的地上部生物量、株高、单株有效角果数、有效分枝数和单株产量好于野生型,其中在中度缺硼(0.25 mg/kg)时L2的蕾薹期地上部干重较野生型提高15.8%,严重缺硼(0.09 mg/kg)时L1和L2单株产量明显高于野生型,中度缺硼时单株产量分别增加3%和10%。研究结果表明转基因植株在缺硼条件下能够较好的生长和结实。
    Boron (B), one of the necessary micronutrients for plant growth, is absorbed and utilized by plants in the form of H3BO3 in soils. In China, especially in the middle and down region of Yangtze River, soil available B deficiency is more severe and the application of B fertilizer has become an essential agronomic measure in B-sensitive crops production, such as Brassica napus and cotton. It was reported that overexpression of B transporter BOR1 in Arabidopsis increased boron transportation and improved seed yield under B-limiting condition.In this research, we transformed BOR1 into Brassica napus cultivar "Weatar 10" and obtained transgenic plants overexpressing BOR1. Then different phenotype identification experiments were carried out to study growth and B efficiency of transgenic plants under low B conditions. The main results were as follows:1. Generation and screening of transgenic plants.13 transforments overexpressing BOR1 gene were obtained By Agrobacterium mediated method. After self-breeding, We got 3,3 and 5 non-segrateing family lines from three transgeneic T2 generation lines, L1, L2, L3, respectively, by Kan screening and PCR identification. Further, we used seeds of the T3 generations to verify that, and seeds of T4 generation ware harvsted.2. Expression of extraneous gene BOR1 and copies identification. Analyzing the expression of BOR1 in different growth stages via RT-PCR showed that transgenic plants could stablely express BOR1 on RNA level. The result from Southern blot analysis indicated that L1 and L2 were double-copys lines, and L3 was a single-copy line.3. Phenotype identification of transgenic plants in seedling stage. Results of hydroponic culture indicated that under severe low-B condition (0.15μM), growth of transgenic plants had advantage over WT, boron concentration and accumulation in shoot of 3 transgenic lines were enhanced to different extent compared with WT. Thereinto, shoot boron concentration of L1 and L2 were almost doubled higher than WT, and shoot boron accumulation of L1, L2 and L3 were 2-3 times higher than WT.4. Phenotype identification of T1 and T3 transgenic plants for whole-growing period. Results of pot trials were as follows:Results of T1 indicated that under moderate B-shortage condition (0.25 mg/kg), silique, seed yield and oil content of L1 and L2 were all increased, and seed yield of L1 is 43% higher than WT. For L3, shoot boron concentration under severe B-shortage condition (0.09 mg/kg) and seed boron concentration under moderate B-shortage condition (0.25 mg/kg) were increased obviously, while seed yield had no difference from WT. Results of T3 indicated that under different boron deficient conditions, shoot yield, plant height, available siliques, available branches and seed yield of L1 and L2 were improved compared with WT. Thereinto, shoot dry weight of L2 in bud stage was 15.8% higher than WT under moderate B-shortage condition (0.25 mg/kg). Seed yield of L1 and L2 were higher than WT under severe B-shortage condition (0.09 mg/kg), and increased by 3% and 10%, respectively, under moderate B-shortage condition (0.25 mg/kg). Results of the experiments indicated that transgenic plants can grow and set seed better than WT when suffering from boron deficiency.
        

超表达BOR1基因提高油菜硼效率的研究

摘要6-7
ABSTRACT7-8
1 前言9-17
    1.1 文献综述10-14
        1.1.1 植物对硼的吸收利用研究进展10-12
        1.1.2 植物硼营养性状的遗传改良研究进展12-14
    1.2 研究意义及内容14-17
        1.2.1 研究意义14-15
        1.2.2 研究内容15-16
        1.2.3 技术路线16-17
2 转基因植株的获得与筛选17-30
    2.1 材料与方法17-23
        2.1.1 转化载体、植物受体及转化过程所用试剂17-18
        2.1.2 农杆菌转化甘蓝型油菜Westar10子叶柄18-19
        2.1.3 阳性植株的分子鉴定19-22
        2.1.4 T_1,T_2,T_3代阳性植株的筛选和纯化22-23
    2.2 结果与分析23-28
        2.2.1 转基因植株的获得23
        2.2.2 转基因植株的筛选和分子鉴定23-28
    2.3 讨论28-30
        2.3.1 油菜的遗传转化28
        2.3.2 抗生素筛选和PCR鉴定28-29
        2.3.3 转基因后代的非孟德尔遗传现象29-30
3 转基因植株对不同硼浓度的反应及其硼的吸收利用30-36
    3.1 材料与方法30
    3.2 结果与分析30-34
        3.2.1 不同硼浓度条件下植株形态和生物量的比较30-32
        3.2.2 不同硼浓度条件下地上部硼含量32-33
        3.2.3 不同硼浓度条件下地上部硼累积量33-34
    3.3 讨论34-36
4 转基因植株全生育期相关性状的考察36-53
    4.1 材料与方法36-37
        4.1.1 材料36
        4.1.2 方法36-37
    4.2 结果与分析37-51
        4.2.1 转基因株系与野生型各生育时期植株长势比较37-41
        4.2.2 转基因植株不同时期不同部位硼含量41-45
        4.2.3 转基因株系成熟期农艺性状和籽粒品质性状的考察45-51
    4.3 讨论51-53
5 总结与展望53-54
参考文献54-62
致谢62
        


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