盾壳霉产黑色素相关基因CmMR1的功能研究

发布时间：2018-03-07 18:56

本文选题：盾壳霉　切入点：CmMR1基因　出处：《华中农业大学》2017年硕士论文　论文类型：学位论文

【摘要】：盾壳霉(Coniothyrium minitans)是一种能寄生核盘菌(Sclerotinia sclerotiorum)菌丝和菌核的重寄生真菌,可用于菌核病的生物防治。盾壳霉菌株ZS-1的T-DNA插入突变体库在前期研究中已构建,本研究以其中一个产黑色素缺陷型突变体ZS-1TN24363为材料进行了深入研究,主要结果如下:1.通过hiTAIL-PCR克隆到了T-DNA插入位点处的侧翼序列,比对分析发现T-DNA插入破坏了一个可能的基因。该基因全长3167 bp,预测含有3个外显子,2个内含子,编码一个含1011个氨基酸的蛋白。该蛋白N端含有一个Zn-clus和Med3的保守结构域,C端含有一个fungal-TF-MHR的保守结构域。进化树分析发现该蛋白与Clohesyomyces aquaticus的转录因子Cmr1蛋白相似性最高,故将该基因命名为CmMR1。2.利用基因敲除技术获得了3个CmMR1基因敲除转化子。敲除转化子和突变体ZS-1TN24363的菌落形态相似,菌落显橙色无黑色素沉积,可产生无色透明的分生孢子器和分生孢子。CmMR1基因互补可使敲除转化子恢复产黑色素表型。基因CmMR1对菌株的生长速率、产孢量、生物学产量、寄生核盘菌菌丝菌和核的能力、对高渗的耐受性等均无明显影响,但CmMR1的敲除使菌丝中ROS含量增多,抗紫外线能力减弱。3.基因CmMR1的敲除使黑色素合成酶基因Cm4HNR、CmSCD、Cm3HNR、CmLAC的表达量下调,证明CmMR1参与调控这些黑色素合成酶基因的表达。上述结果表明,基因CmMR1不影响盾壳霉的生长、产孢和寄生,但是与黑色素的生物合成有关。
[Abstract]:C.minitans (Coniothyrium minitans) is a parasitic Sclerotinia (Sclerotinia sclerotiorum) hyperparasitic fungi mycelium and sclerotium, can be used for biological control of Sclerotinia sclerotiorum. C.minitans ZS-1 T-DNA insertion mutant library has been constructed in the previous study, this research has conducted the thorough research to a black pigmented mutant ZS-1TN24363 in the material, the main results are as follows: 1. flanking sequence was cloned by hiTAIL-PCR to the T-DNA insertion site of the alignment analysis showed that the T-DNA insertion damaged a possible gene. The gene was 3167 BP. The forecast contains 3 exons and 2 introns, encoding a protein of 1011 amino acids conserved domain. The protein contains a Zn-clus N end and Med3 end, conserved domain of C contains a fungal-TF-MHR. Phylogenetic analysis showed that the transcription factor C protein and Clohesyomyces aquaticus MR1 protein is the highest similarity, so the gene was named CmMR1.2. using gene knockout technology obtained 3 CmMR1 gene knockout transformants. Knockout transformants and colony morphology mutants are similar to that of ZS-1TN24363, no significant colony orange melanin deposition, can produce colorless and transparent conidia and conidia of.CmMR1 the complementary gene knockout transformants restored black pigmented phenotype. The growth rate of CmMR1 gene of strain, sporulation, biological yield, parasitic Sclerotinia mycelium and nuclear power, had no significant effect on osmotic tolerance, but CmMR1 knockout ROS content in mycelia increased resistance ultraviolet CmMR1 knockout attenuated.3. melanin synthase gene Cm4HNR, CmSCD, Cm3HNR, CmLAC expression down regulated expression of CmMR1 in the regulation of melanin biosynthesis gene. The results showed that the CmMR1 gene does not affect the shield shell The growth, sporulation, and parasitism of molds, but related to the biosynthesis of melanin.

【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S476

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