【作者】 刘婷；

【导师】 王惠； 刘在民；

【作者基本信息】 沈阳农业大学 ， 生物工程（专业学位）， 2016， 硕士

【摘要】 大豆胞囊线虫(Soybean Cyst Nematode,简称SCN)病是世界上危害大豆生产最严重的病害之一,因此,对SCN的研究一直受到世界各地政府及科研人员的重视。由于农业和化学等防治措施存在诸多不足,所以抗线虫基因工程成为目前研究热点,将克隆的一个或多个抗性基因载体转化到目的植物中,使植物产生更强的长久抗性,从而达到防治线虫的目的。在大豆基因组上,发现肌醇加氧酶基因,命名为GmMIOX,通过转录组测序发现该基因在SCN侵染前后出现明显表达差异,推测该基因可能与SCN的抗性有关。但目前还没有大豆MIOX基因的相关研究报道,大多研究动物肌醇加氧酶基因。故本研究旨在研究GmMIOX基因在大豆胞囊线虫三号生理小种(SCN3)侵染下是否起到抗性作用,并验证其功能。本研究选择抗、感病两种大豆品种灰皮支黑豆和辽豆15,通过RT-PCR技术筛选出GmMIOX基因家族中可能起抗性作用的一个基因作为主要研究对象,并通过实时荧光定量PCR技术检测其在不同时期接种SCN3前后表达量的变化并构建过表达载体。研究主要结果如下：1.发现SCN3可以侵入抗病品种灰皮支黑豆根内,但灰皮支黑豆中SCN3侵入的数目少于辽豆15,且在灰皮支黑豆中三龄幼虫和成虫数量比辽豆15少,说明灰皮支黑豆对线虫具有抗繁殖作用。2.通过RT-PCR检测,发现GmMIOX4基因在辽豆15和灰皮支黑豆中均得到特异性扩增目的产物,条带明显,且差异表达明显,推断其可能对SCN3起抗性作用,故本研究最终选用GmMIOX4基因。3.利用实时荧光定量PCR方法明确了GmMIOX4基因在不同时期、不同品种接种SCN3前后表达量的变化规律。在无SCN3侵染下,辽豆15根内的GmMIOX4基因相对表达量呈逐渐下降趋势,而灰皮支黑豆根内的GmMIOX4基因相对表达量无明显规律,在15 dpi (days post inoculation)时出现相对表达量的最高点；在SCN3侵染下,辽豆15根内的GmMIOX4基因的相对表达量非常低,直到接种25 dpi时,GmMIOX4基因的相对表达量升高,而灰皮支黑豆根内的GmMIOX4基因在接种SCN3前期的相对表达量较低,在接种10 dpi时GmMIOX4基因的相对表达量达到最高,说明很可能在10 dpi时,GmMIOX4基因起抗性作用,使合胞体结构发生改变,线虫不能依靠合胞体吸取足够生长、发育的营养,从而使灰皮支黑豆达到抑制线虫发育的作用,灰皮支黑豆根内成虫数量减少,起到抗病效果,或在SCN3穿过根表皮的迁移阶段,使其在细胞内向维管束移动过程中受阻,从而起到抗病效果。4.从抗病品种灰皮支黑豆中通过RT-PCR方法得到GmMIOX4基因的CDS区,克隆到pGEM-T easy载体中,再以得到的GmMIOX4序列为材料,在该序列两端加上双酶切位点,经过双酶切后与植物双元表达载体pCAMBIA1303连接,构建pCAM-MIOX4过表达载体,使GmMIOX4基因高效稳定表达,后续可将其转化到植物细胞内,在进行SCN3侵染率的测定,因此,该载体的成功构建为进一步研究GmMIOX4基因功能奠定了坚实的基础。更多还原

【Abstract】 The Soybean Cyst Nematode disease is one of the serious disease that decreasing the production of soybean in the world today. Therefore, the study on the Soybean Cyst Nematode disease has been gaining increasing attention in the group of governments and researchers. Agricultural and chemical control measures have many problems, so nematode resistant base for genetic engineering become hotspot of research at present, cloned one or a plurality of resistance gene vectors transformed into plant, which enables the plant to produce a stronger, more durable resistance, in order to achieve a defense.Inositol oxygenase gene was found in the soybean genome, which is named for GmMIOX, by transcriptome sequencing, found that the gene before and after the Soybean Cyst Nematode infection appear significantly different expression, suggesting that the gene may be associated with soybean to Soybean Cyst Nematode resistance. However, there is no relevant studies has been reported in the field of soybean’s MIOX gene, much more studies of MIOX gene are focused on the research field of animals. Therefore, the aim in this study was to investigate whether the gene plays GmMIOX resistance in infection under SCN3, and to verify its function.There are two kinds of soybean in the research of resistant and sense of disease, Huipizhi Heidou and Liaodou 15, using RT-PCR screened GmMIOX gene family may exert resistant effect on a gene as the main object of study and by real-time quantitative PCR detecting its before and after expression changes in different periods of SCN3 inoculated and constructed over expression vector. The main results are as follows:1.The results showed that the nematode can invade the resistant varieties Huipizhi Heidou, but the number of nematode invasion huipizhi Heidou is less than Liaodou 15, in Huipizhi Heidou third instar larvae and adult numbers significantly less than Liaodou 15 that Huipizhi Heidou to nematodes is resistant to the development of anti-propagation effects.2.Detected by RT-PCR, it is found that GmMIOX4 gene in Liaodou 15 and Huipizhi Heidou are specific amplified product with obvious strip, and differences in the expression, it can be inferred that the possible effect of resistance to Soybean Cyst Nematode disease, so we choose the final selection of gene GmMIOX4.3.By real-time quantitative PCR method identified GmM10X4 gene expression changes in different periods, the amount of different varieties before and after inoculation of SCN3. In the absence of SCN3 infection, the relative expression of Liaodou 15 gene of GmMIOX4 in the root volume decreased gradually, while GmMIOX4 based Huipizhi Heidou root for relative expression without obvious the regulation, appeared in the 15 dpi when the relative expression amount reached highest point; under SCN3 infection, Liaodou 15 roots in the relative expression of GmMIOX4 gene is very low, until 25 dpi after inoculation, the relative expression of GmMIOX4 gene and GmMIOX4 gene increased, Huipizhi Heidou root inoculation of previous SCN3 relative expression low relative expression of GmMIOX4 gene in inoculated when 10 dpi reached the highest level, that is likely to be in the 10 dpi, the GmM10X4 gene plays resistance role, make the syncytial structure changed, nematodes can not rely on the syncytium absorb enough growth, the development of the nutrition, so that Huipizhi Heidou to suppress the role of nematode development, reduce the Huipizhi Heidou root number of adult, effect of resistance, or through the root epidermis of stage migration in the Soybean Cyst Nematode, which is blocked in the cells within the vascular bundles in the process of moving, play a disease resistant effect.4. From the Huipizhi Heidou by RT-PCR in CDS zone obtained GmMIOX4 gene, cloned into pGEM-T easy vector. The resulting sequence GmMIOX4 as materials, in the sequence ends with double enzyme cutting sites. After double enzyme digestion and plant binary expression vector pCAMBIA1303 connection, construction pCAM-MIOX4 over expression vector and the GmMOX4 gene, stable expression, which can be transformed into the plant cells, in the determination of SCN3 infection rate, therefore, to build a successful carrier for further study of its function and lay a solid foundation.更多还原