中间偃麦草天冬氨酸蛋白酶基因TiAP1的功能分析

发布时间：2018-06-12 21:11

本文选题：白粉病 + 赤霉病　；参考：《山东农业大学》2017年硕士论文

【摘要】：小麦是我国种植最广泛的农作物之一,全球大约1/3的人口以小麦为主食。但是,小麦白粉病和赤霉病一直是影响小麦高产、稳产的重要限制因素。因此,挖掘新的抗病基因,培育出含有多个抗病基因聚合的抗病小麦品种是目前亟待解决的问题。在前期研究中,从小偃麦异附加系SN6306(高感白粉的小麦品种烟农15与中间偃麦草杂交后代)中克隆到了一个与白粉病抗性相关的天冬氨酸蛋白酶基因TiAP1。本研究以天冬氨酸蛋白酶基因TiAP1为研究对象,对TiAP1基因的功能及作用机制进行分析,主要结果如下:(1)TiAP1蛋白的生物信息学分析目的蛋白为天冬氨酸蛋白酶,通过对跨膜螺旋进行预测,发现目的蛋白没有跨膜结构,因此不是跨膜蛋白。同时对蛋白的许多物理化学特性进行了分析,目的蛋白含有506个氨基酸,等电点为5.6,含有信号肽,信号肽的切割位点位于第16和17个氨基酸之间。除此之外还对蛋白的二级结构和三级结构进行了预测,该蛋白的二级结构中延伸链和无规则卷曲所占的比例较高,三级结构构建的模型比较稳定。(2)原核表达系统进行蛋白质的功能分析利用本实验室现有的pET-28a载体与密码子优化后的目的序列构建原核表达载体pET-AP,重组质粒转入BL21中,加入IPTG诱导大肠杆菌表达,37℃诱导过夜,利用SDS-PAGE电泳,在45KD附近出现了目的条带。利用Western blot对目的蛋白进行检测,证明诱导表达成功。利用亲和层析,在将目的蛋白分离纯化出来的过程中,发现目的蛋白表达是以包涵体的形式。将目的蛋白纯化复性后进行白粉病菌孢子和赤霉病菌孢子的萌发试验,结果发现目的蛋白会抑制白粉病菌和赤霉病菌的孢子的萌发,(3)TiAP1蛋白作用机制分析通过分生孢子萌发实验,发现TiAP1蛋白可以影响白粉病菌和禾谷镰刀菌分生孢子的萌发。为了进一步确定目的蛋白的作用机制,将禾谷镰刀菌的菌丝和分生孢子分别放入蛋白储存液、低浓度(0.146mg/ml)蛋白、高浓度(0.291mg/ml)蛋白溶液中孵育,孵育结束后,加入荧光染料SYTOX Green,观察。结果发现在蛋白储存液中孵育之后的菌丝和分生孢子加入荧光染料之后也没有荧光产生;经过低浓度蛋白溶液孵育后的菌丝和孢子,在显微镜下菌丝和分生孢子带有微弱的荧光;高浓度的蛋白溶液中荧光增强。以上结果说明TiAP1蛋白可以增加细胞膜的通透性。(4)转基因小麦对白粉病菌的抗性鉴定本实验室前期研究中得到了转基因BobWhite小麦,进行小麦白粉病菌E09的苗期抗病性鉴定,发现转基因小麦植株叶片上的孢子数明显少于对照。说明TiAP1基因可能会延缓白粉病的侵染速度。此外,为了进一步探究TiAP1基因可能参与的抗白粉病的途径,通过白粉病菌诱导,转基因小麦中茉莉酸甲酯的标记基因OPDA、PR10、COL1标记基因跟0h和对照相比,表达量明显上调,其中PR10的表达量上调最多,是0h的20倍左右。这些结果表明TiAP1基因可能参与了茉莉酸激素调节途径。(5)转基因拟南芥对Pst DC3000响应将之前构建好的拟南芥表达载体pRI-AP1,基因转入拟南芥中后,经过筛选,得到转基因拟南芥。随后,将Pst DC3000菌喷洒野生型拟南芥,收集处理3d后的叶片进行苯胺蓝、台盼蓝、DAB染色,用以检测叶片中胼胝质的积累、活性氧的测定以及会死细胞的含量,结果表明转基因拟南芥中胼胝质、坏死细胞、活性氧积累的要比野生型多。这些结果表明TiAP1基因可以增强拟南芥对Pst DC3000的抗性。
[Abstract]:Wheat is one of the most widely cultivated crops in China, and the population of about 1/3 is staple food in the world. But wheat powdery mildew and scab are the important limiting factors for high yield and stable yield of wheat. Therefore, it is urgent to excavate new disease resistant genes and cultivate resistant wheat varieties with multiple disease resistant bases. In the previous study, an aspartic protease gene TiAP1. related to powdery mildew resistance was cloned from the progeny of SN6306 (high sensitivity white wheat cultivar tobacco grower 15 and intermedium Agropyron intermedium). The study on aspartic acid protease gene TiAP1 as the research object and the function and action machine of TiAP1 gene The main results are as follows: (1) the target protein of the bioinformatics analysis of TiAP1 protein is aspartic protease. Through the prediction of the transmembrane helix, it is found that the target protein has no transmembrane structure and therefore is not a transmembrane protein. At the same time, many physicochemical properties of the protein are analyzed, and the target protein contains 506 amino acids. The isoelectric point is 5.6, contains the signal peptide, the cutting site of the signal peptide is located between sixteenth and seventeenth amino acids. In addition, the two structure and the three structure of the protein are predicted. The proportion of the extension chain and the irregular curl in the two structure of the protein is higher, and the model of the three grade structure is stable. (2) the prokaryotic expression system (2) The function analysis of protein was used to construct the prokaryotic expression vector pET-AP with the sequence of pET-28a vector and codon optimized in our laboratory. The recombinant plasmid was transferred into BL21, the expression of Escherichia coli was induced by IPTG, the night was induced at 37 C, and the target band was found near 45KD by SDS-PAGE electrophoresis. The purpose was Western blot for the purpose. The protein was detected and proved to be successful. In the process of separating and purifying the target protein by affinity chromatography, the expression of the target protein was found in the form of inclusion body. After purify the refolding of the target protein, the spore germination of powdery mildew and scab was tested. The results showed that the target protein could inhibit the powdery mildew pathogen. The germination of spores of the spore of scab, (3) the mechanism of the action of TiAP1 protein was analyzed through the germination experiment of conidia. It was found that TiAP1 protein could affect the germination of the conidia of Fusarium graminearum and Fusarium graminearum. In order to further determine the mechanism of the action of the target protein, the hypha and conidium of Fusarium graminearum were put into the protein storage solution, respectively. After incubation of low concentration (0.146mg/ml) protein and high concentration (0.291mg/ml) protein solution, the fluorescent dye SYTOX Green was added after incubation. The results showed that the hypha and conidium after incubation in the protein storage solution were not produced by fluorescent dye; the hypha and spores after incubation with the low concentration protein solution were found. Mycelium and conidia were weak fluorescence under microscope; fluorescence enhancement in high concentration protein solution. The results indicated that TiAP1 protein could increase the permeability of cell membrane. (4) the resistance identification of transgenic wheat to powdery mildew fungus was studied in the previous study of transgenic wheat with BobWhite wheat and E09 of wheat powdery mildew. It was found that the number of spores on the leaves of the transgenic wheat was obviously less than that of the control. The TiAP1 gene might delay the infection rate of powdery mildew. In addition, in order to further explore the possible way to resist powdery mildew of the TiAP1 gene, the marker gene OPDA, PR of the methyl jasmonate in transgenic wheat was induced by powdery mildew. 10, the expression of COL1 marker gene was up to rise compared with 0h and control, in which the expression of PR10 was up to 20 times that of 0h. These results suggest that the TiAP1 gene may be involved in the regulatory pathway of jasmonate. (5) transgenic Arabidopsis thaliana response to Pst DC3000 will be constructed as the Arabidopsis expression vector pRI-AP1, and the gene is transferred into the south of the south. After screening, the transgenic Arabidopsis thaliana was obtained through screening. Then, Pst DC3000 bacteria were sprayed with wild Arabidopsis, and the leaves were collected and treated with 3d blue, trypan blue and DAB. The accumulation of callose, the determination of reactive oxygen species and the content of dead cells were detected in the leaves. The results showed callose and necrotic cells in transgenic Arabidopsis. The results showed that TiAP1 gene could enhance the resistance of Arabidopsis thaliana to Pst DC3000.
【学位授予单位】：山东农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S435.121.4

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