【作者】 王超；

【导师】 王惠；

【作者基本信息】 沈阳农业大学 ， 生物化学与分子生物学， 2019， 硕士

【摘要】 大豆作为我国的主要农作物,长年受到生物或非生物胁迫,前人关于盐碱、干旱等非生物胁迫已有颇多的报道,但有关生物胁迫的研究较少。大豆胞囊线虫(Soybean Cyst Nematode,简称:SCN)病是主要生物胁迫病害之一,侵染植株严重。因此,如何防治SCN已逐渐成为科学家研究的重点。传统的防治方法为轮作或者种植抗病品种,但传统方法容易导致线虫产生适应性,因此随着基因工程技术的发展,筛选抗性基因并培育抗性植株成为主要防治手段并取得成效。肌醇磷酸合成酶(MIPS)催化葡萄糖-6-磷酸(G-6-P)生成肌醇-1-磷酸,此步骤为真核生物肌醇合成的限速步骤。肌醇参与植物体中多种代谢途径,并且与植物细胞壁的生物合成密切相关,肌醇可以通过自身氧化代谢途径生成参与细胞壁合成的多糖:木聚糖以及果胶。由此推断,调控MIPS基因的表达水平可增强大豆细胞壁的合成用于抵抗线虫的侵染。在大豆基因组上发现MIPS基因,命名为GmMIPS,其基因家族包括四个基因。本试验选择抗病品种灰皮支黑豆、哈尔滨小黑豆、小粒黑豆以及感病品种辽豆15,分别在SCN三号生理小种(SCN3)侵染后的不同时间段取样,提取RNA并反转录为cDNA,利用荧光定量PCR检测SCN侵染后GmMIPS1和GmMIPS2的表达分析,构建瞬时表达载体以及GmMIPS基因编码蛋白的亚细胞定位,为后续功能研究奠定基础。主要研究结果如下:1.通过生物信息学检测GmMIPS1和GmMIPS2基因长度为1533bp,编码511个氨基酸,二者均为稳定性亲水蛋白质,分别具有38和39个磷酸化位点,具有较强的磷酸化能力。MIPS基因属于肌醇合成的上游基因,参与肌醇生物合成并属于肌醇磷酸化途径。在常见的15种植株中,与GmMIPS亲缘关系最近的是菜豆和绿豆,亲缘关系最远的是玉米和小麦。2.通过酸性品红染色发现SCN3可以侵染抗感病大豆植株根部并定植于此,继续发育为三龄、四龄幼虫,同时发现SCN3侵染感病植株的数量明显多于抗病品种,说明抗病植株存在某种抗性机制可以抵抗线虫侵染。3.利用反转录PCR(RT-PCR)技术,分别从大豆抗病品种灰皮支黑豆、哈尔滨小黑豆、小粒黑豆以及感病大豆品种辽豆15中克隆出长度为1533bp的GmMIPS1和GmMIPS2基因。利用实时荧光定量PCR(qPCR)技术明确了在SCN3胁迫下,抗感大豆品种中GmMIPS1和GmMIPS2基因的表达量变化规律。结果发现:三种大豆抗病品种在SCN3侵染10 dpi(days post inoculation)后GmMIPS1和GmMIPS2的表达量均上升并高于在感病品种辽豆15中的表达量,说明目的基因在10 dpi发挥抗性作用来抵御线虫的发育。4.选取抗病大豆品种中表现显著差异的实验组cDNA为模板,成功构建细菌克隆载体pMD19-GmMIPS1和pMD19-GmMIPS2,随即与融合过表达载体pCAMBIA1303连接,成功构建植物过表达载体pCAMBIA1303-GmMIPS1和pCAMBIA1303-GmMIPS2。5.通过农杆菌介导的烟草瞬时表达系统,在本生烟叶片中瞬时表达GmMIPS1和GmMIPS2蛋白。利用激光共聚焦显微镜观察荧光蛋白GFP的位置,试验结果表明:阳性对照组荧光均匀分布在细胞上,阴性对照中无荧光,GmMIPS1蛋白和GmMIPS2蛋白荧光主要分布在细胞膜或者细胞壁上。更多还原

【Abstract】 Soybean is an important crop in China that subjected to biotic and abiotic stress all year round,several reports have been studied about abiotic stresses such as salinity and srought,but few biotic stress.Soybean Cyst Nematode(SCN)is the main biotic stress diseases.Therefore,preventing and controling the demage of SCN has gradually become the focus of scientists‘ research.The traditional control methods is crop rotation and planted resistant varieties,but the traditional metods is causing the nematodes provide adaptation.With the development of fenetic engineering technology,screening resistant genes and cultivating resistant plants have become the main prevention and control methods,and achieved results.Myo-inositol phosphate synthase catalyzes the rate-limiting step in synthesis of inositol-1-phosphate by glucose-6-phosphate(G-6-P).Inositol is the main storage form phosphorus in plants and inositol can produce polysaccharides involved in cell wall synthesis through auto-oxidative metabolic pathways,mainly xylan and pectin.Therefore,increased the expression of MIPS gene can enhance the resistance of soybean plants to nematodes.The MIPS gene was found in soybean genome that named Gm MIPS.MIPS gene family have four members.In this study,the resistant cultivars Huipizhi Heidou,Harbin Xiao heidou and Xiaoli Heidou and the susceptible cultivar Liaodou 15 were infected by SCN3,RNA was extracted at differernt time.This study aim is to investigate the expression analysis of the candidate genes Gm MIPS1 and Gm MIPS2 under infected by SCN3,construct the transient expression vector and detect the subcellular localization of MIPS protein for the preliminary study.The main research results include:1.The length of Gm MIPS1 and Gm MIPS2 is 1533 bp and encoding 511 amino acids,both of which are stable hydrophilic amino acids,with 38 and 39 phosphorylation sites and strong phosphorylation ability.MIPS belongs to the upstream gene of inositol synthesis,involved in inositol biosynthesis and belongs to the inositol phosphorylation pathway.Among the 15 common plants,the closest relationship with Gm MIPS is the bean and mung bean,and the farthest relationship is corn and wheat.2.The results showed that SCN3 can infect the roots of soybean and colonize in it by acid fuchsin staining and continue to develop in third and forth instar larvae.Meanwhile,it found that the number of SCN3 infected susceptible plants was more than that of resistant varieties.3.The Gm MIPS1 and Gm MIPS2 genes from the the resistant cultivars Huipizhi heidou,Harbin Xiao heidou and Xiaoli Heidou and a susceptible cultivar Liaodou 15 were cloned by RT-PCR that the 1533 bp length.By real-time quantitative PCR method identified Gm MIPS1 and Gm MIPS2 genes‘ regulation under the stress of SCN3.The results showed that the expression of Gm MIPS1 and Gm MIPS2 in three resistant cultivars were increased and higher than the susceptible cultivar Liaodou 15 at 10 dpi,indicating that the Gm MIPS genes exerted resistance at 10 dpi and inhibited the development of SCN.4.Selected the c DNA from the experimental resistant soybean variety,constructed p MD19-Gm MIPS1 and p MD19-Gm MIPS2.After double enzyme digestion and plant binary expression vector p CAMBIA1303 connection,then constructed p CAMBIA 1303-Gm MIPS1 and p CAMBIA130-Gm MIPS2.5.Gm MIPS1 and Gm MIPS2 were expressed in the leaves of tobacco by agrobacterium-mediated tobacco transient expression system.Observed the fluorescent protein GFP by laser confocal microscopy.The subcellular localization result showed that Gm MIPS1 and Gm MIPS2 protein was located on the cytomembrane or cell wall.更多还原