本文選題：白粉病 + 赤霉病�。� 參考：《山東農(nóng)業(yè)大學(xué)》2017年碩士論文

【摘要】：小麥?zhǔn)俏覈N植最廣泛的農(nóng)作物之一,全球大約1/3的人口以小麥為主食。但是,小麥白粉病和赤霉病一直是影響小麥高產(chǎn)、穩(wěn)產(chǎn)的重要限制因素。因此,挖掘新的抗病基因,培育出含有多個(gè)抗病基因聚合的抗病小麥品種是目前亟待解決的問題。在前期研究中,從小偃麥異附加系SN6306(高感白粉的小麥品種煙農(nóng)15與中間偃麥草雜交后代)中克隆到了一個(gè)與白粉病抗性相關(guān)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為研究對象,對TiAP1基因的功能及作用機(jī)制進(jìn)行分析,主要結(jié)果如下:(1)TiAP1蛋白的生物信息學(xué)分析目的蛋白為天冬氨酸蛋白酶,通過對跨膜螺旋進(jìn)行預(yù)測,發(fā)現(xiàn)目的蛋白沒有跨膜結(jié)構(gòu),因此不是跨膜蛋白。同時(shí)對蛋白的許多物理化學(xué)特性進(jìn)行了分析,目的蛋白含有506個(gè)氨基酸,等電點(diǎn)為5.6,含有信號肽,信號肽的切割位點(diǎn)位于第16和17個(gè)氨基酸之間。除此之外還對蛋白的二級結(jié)構(gòu)和三級結(jié)構(gòu)進(jìn)行了預(yù)測,該蛋白的二級結(jié)構(gòu)中延伸鏈和無規(guī)則卷曲所占的比例較高,三級結(jié)構(gòu)構(gòu)建的模型比較穩(wěn)定。(2)原核表達(dá)系統(tǒng)進(jìn)行蛋白質(zhì)的功能分析利用本實(shí)驗(yàn)室現(xiàn)有的pET-28a載體與密碼子優(yōu)化后的目的序列構(gòu)建原核表達(dá)載體pET-AP,重組質(zhì)粒轉(zhuǎn)入BL21中,加入IPTG誘導(dǎo)大腸桿菌表達(dá),37℃誘導(dǎo)過夜,利用SDS-PAGE電泳,在45KD附近出現(xiàn)了目的條帶。利用Western blot對目的蛋白進(jìn)行檢測,證明誘導(dǎo)表達(dá)成功。利用親和層析,在將目的蛋白分離純化出來的過程中,發(fā)現(xiàn)目的蛋白表達(dá)是以包涵體的形式。將目的蛋白純化復(fù)性后進(jìn)行白粉病菌孢子和赤霉病菌孢子的萌發(fā)試驗(yàn),結(jié)果發(fā)現(xiàn)目的蛋白會抑制白粉病菌和赤霉病菌的孢子的萌發(fā),(3)TiAP1蛋白作用機(jī)制分析通過分生孢子萌發(fā)實(shí)驗(yàn),發(fā)現(xiàn)TiAP1蛋白可以影響白粉病菌和禾谷鐮刀菌分生孢子的萌發(fā)。為了進(jìn)一步確定目的蛋白的作用機(jī)制,將禾谷鐮刀菌的菌絲和分生孢子分別放入蛋白儲存液、低濃度(0.146mg/ml)蛋白、高濃度(0.291mg/ml)蛋白溶液中孵育,孵育結(jié)束后,加入熒光染料SYTOX Green,觀察。結(jié)果發(fā)現(xiàn)在蛋白儲存液中孵育之后的菌絲和分生孢子加入熒光染料之后也沒有熒光產(chǎn)生;經(jīng)過低濃度蛋白溶液孵育后的菌絲和孢子,在顯微鏡下菌絲和分生孢子帶有微弱的熒光;高濃度的蛋白溶液中熒光增強(qiáng)。以上結(jié)果說明TiAP1蛋白可以增加細(xì)胞膜的通透性。(4)轉(zhuǎn)基因小麥對白粉病菌的抗性鑒定本實(shí)驗(yàn)室前期研究中得到了轉(zhuǎn)基因BobWhite小麥,進(jìn)行小麥白粉病菌E09的苗期抗病性鑒定,發(fā)現(xiàn)轉(zhuǎn)基因小麥植株葉片上的孢子數(shù)明顯少于對照。說明TiAP1基因可能會延緩白粉病的侵染速度。此外,為了進(jìn)一步探究TiAP1基因可能參與的抗白粉病的途徑,通過白粉病菌誘導(dǎo),轉(zhuǎn)基因小麥中茉莉酸甲酯的標(biāo)記基因OPDA、PR10、COL1標(biāo)記基因跟0h和對照相比,表達(dá)量明顯上調(diào),其中PR10的表達(dá)量上調(diào)最多,是0h的20倍左右。這些結(jié)果表明TiAP1基因可能參與了茉莉酸激素調(diào)節(jié)途徑。(5)轉(zhuǎn)基因擬南芥對Pst DC3000響應(yīng)將之前構(gòu)建好的擬南芥表達(dá)載體pRI-AP1,基因轉(zhuǎn)入擬南芥中后,經(jīng)過篩選,得到轉(zhuǎn)基因擬南芥。隨后,將Pst DC3000菌噴灑野生型擬南芥,收集處理3d后的葉片進(jìn)行苯胺藍(lán)、臺盼藍(lán)、DAB染色,用以檢測葉片中胼胝質(zhì)的積累、活性氧的測定以及會死細(xì)胞的含量,結(jié)果表明轉(zhuǎn)基因擬南芥中胼胝質(zhì)、壞死細(xì)胞、活性氧積累的要比野生型多。這些結(jié)果表明TiAP1基因可以增強(qiáng)擬南芥對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.
【學(xué)位授予單位】：山東農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S435.121.4

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 張安存;朱祥林;滕志英;陳文軍;崔金登;韓淮林;;不同藥劑防治小麥赤霉病篩選研究試驗(yàn)[J];種子科技;2016年12期

2 龐海龍;;小麥赤霉病發(fā)病癥狀與防治方法[J];農(nóng)民致富之友;2016年23期

3 丁麗娜;楊國興;;植物抗病機(jī)制及信號轉(zhuǎn)導(dǎo)的研究進(jìn)展[J];生物技術(shù)通報(bào);2016年10期

4 楊銀;高志勇;;植物抗病機(jī)制的研究進(jìn)展[J];科教導(dǎo)刊(中旬刊);2016年09期

5 楊美娟;黃坤艷;韓慶典;;小麥白粉病及其抗性研究進(jìn)展[J];分子植物育種;2016年05期

6 劉易科;佟漢文;朱展望;陳泠;鄒娟;張宇慶;高春保;;小麥赤霉病抗性機(jī)理研究進(jìn)展[J];中國農(nóng)業(yè)科學(xué);2016年08期

7 王亞銳;吳燕;;植物天冬氨酸蛋白酶的功能研究進(jìn)展[J];生命科學(xué);2016年03期

8 葛偉娜;李超;張家琦;張嵐;王振怡;;植物天冬氨酸蛋白酶的研究進(jìn)展[J];生物技術(shù)通報(bào);2016年01期

9 安霞;張海軍;蔣方山;呂連杰;陳軍;鞏素霞;崔麗艷;;小麥赤霉病的發(fā)生與防治[J];現(xiàn)代農(nóng)業(yè)科技;2015年22期

10 高紅云;楊習(xí)文;賀德先;牛吉山;王晨陽;吳寅;;小麥白粉病對籽粒產(chǎn)量和品質(zhì)的影響及防控途徑[J];山東農(nóng)業(yè)科學(xué);2015年10期

相關(guān)博士學(xué)位論文 前2條

1 趙蘭飛;小麥赤霉�、蛐涂剐詸C(jī)理研究及相關(guān)基因的功能鑒定[D];山東農(nóng)業(yè)大學(xué);2016年

2 王曉清;擬南芥抗病突變體cpr30的蛋白質(zhì)組學(xué)研究及抗病機(jī)制初探[D];中國農(nóng)業(yè)大學(xué);2014年

相關(guān)碩士學(xué)位論文 前2條

1 宋靜;中間偃麥草抗白粉病相關(guān)基因TiAsp的克隆與功能研究[D];山東農(nóng)業(yè)大學(xué);2015年

2 巨嵐;小麥生理型雄性不育系中天冬氨酸蛋白酶與絨氈層代謝的相關(guān)性分析[D];西北農(nóng)林科技大學(xué);2015年

，

本文編號：2011103