【摘要】：盾殼霉是植物病原真菌核盤菌的重寄生菌,在菌核病的生物防治中具有舉足輕重的作用。在前期,實(shí)驗(yàn)室通過農(nóng)桿菌介導(dǎo)的轉(zhuǎn)化(ATMT)獲得了盾殼霉的T-DNA插入突變體庫。通過篩選發(fā)現(xiàn)突變體ZS-1TN4685表型異常。通過克隆T-DNA插入位點(diǎn)的側(cè)翼序列,并將其在盾殼霉基因組中進(jìn)行比對(duì),發(fā)現(xiàn)破壞了基因Cm_03805。本研究以ZS-1TN4685為研究材料,并對(duì)基因Cm_03805的功能進(jìn)行了初步研究,研究結(jié)果如下:1.與野生型ZS-1相比,突變體ZS-1TN4685菌落形態(tài)不規(guī)則,生長速率下降。在生長后期,菌落生長停止,且不能長滿整個(gè)培養(yǎng)皿。在顯微鏡下觀察發(fā)現(xiàn)菌絲尖端彎曲、分枝增多。此外,突變體產(chǎn)孢量下降,但孢子寄生菌核的能力無顯著差別。2.將基因Cm_03805在NCBI上進(jìn)行比對(duì)發(fā)現(xiàn)該基因含有端粒酶RNA結(jié)合結(jié)構(gòu)域和端粒酶逆轉(zhuǎn)錄酶特異性結(jié)構(gòu)域,故將該基因命名為CmTERT�？寺≡摶虬l(fā)現(xiàn)其全長為4155 bp,含有10個(gè)外顯子,9個(gè)內(nèi)含子,編碼1168個(gè)氨基酸。3.通過本地blast比對(duì),發(fā)現(xiàn)基因CmTERT在盾殼霉基因組中為單拷貝。進(jìn)化分析發(fā)現(xiàn)該蛋白與番茄匍柄霉的端粒酶逆轉(zhuǎn)錄酶具有較高的同源性。對(duì)CmTERT的表達(dá)動(dòng)態(tài)分析發(fā)現(xiàn)該基因在分生孢子器形成起始期表達(dá)量高。4.根據(jù)同源重組的原理,對(duì)CmTERT進(jìn)行了基因敲除,獲得了4株該基因的敲除轉(zhuǎn)化子。與野生型ZS-1相比,基因CmTERT的敲除突變體生長速率降低,菌絲尖端彎曲、分枝增多,生物學(xué)產(chǎn)量降低,菌絲ROS含量升高,產(chǎn)孢量下降,但孢子寄生菌核的能力無明顯變化。這些特征與突變體ZS-1TN4685基本一致。以上研究結(jié)果表明,基因CmTERT參與調(diào)控盾殼霉的生長和產(chǎn)孢量,但不影響孢子的寄生能力,這對(duì)于深入理解盾殼霉的生長發(fā)育具有重要意義。
[Abstract]:As a hyperparasite of Sclerotinia sclerotiorum, it plays an important role in the biological control of Sclerotinia sclerotiorum (Sclerotinia sclerotiorum). In the early stage, the T-DNA insertion mutant library was obtained by Agrobacterium tumefaciens mediated transformation of (ATMT). The mutant ZS-1TN4685 phenotype was found to be abnormal by screening. By cloning the flanking sequence of the insertion site of T-DNA, and comparing it with the genome of C. capsidii, it was found that the gene Cm_03805. was destroyed. In this study, ZS-1TN4685 was used as the research material and the function of gene Cm_03805 was preliminarily studied. The results are as follows: 1. Compared with wild type ZS-1, the mutant ZS-1TN4685 colony was irregular in shape and decreased in growth rate. At the late stage of growth, colony growth stopped and the whole dish could not grow. The mycelium tip was curved and branched under microscope. In addition, the spore production of the mutant decreased, but there was no significant difference in the ability of spore parasite. The gene Cm_03805 was compared on NCBI. It was found that the gene contained telomerase RNA binding domain and telomerase reverse transcriptase specific domain, so the gene was named CmTERT.. The total length of the gene was 4155 bp, with 10 exons and 9 introns, encoding 1168 amino acids. The gene CmTERT was found to be a single copy in the genome of Trichoderma auratus by local blast comparison. Evolutionary analysis showed that the protein had high homology with the telomerase reverse transcriptase of Tomato. Dynamic analysis of the expression of CmTERT showed that the gene was highly expressed in the initial stage of conidial organ formation. According to the principle of homologous recombination, CmTERT gene knockout was carried out, and four knockout transformants were obtained. Compared with wild-type ZS-1, knockout mutants of gene CmTERT decreased in growth rate, mycelium tip bent, branching increased, biological yield decreased, hyphal ROS content increased and sporulation decreased, but the ability of spore parasite sclerotia did not change significantly. These characteristics are basically consistent with the mutant ZS-1TN4685. These results suggest that the gene CmTERT is involved in the regulation of the growth and sporulation of C. capsidii, but does not affect the parasitic ability of the spores, which is of great significance for the further understanding of the growth and development of the fungus.
【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：S476

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