| Alternaria alternata (Fr.) Keissler, a natural pathogenic filamentous fungi, was isolated from worst weed Croftonweed (Eupatorium adenophorum Spreng.) and has potentiality been evaluated as an alternative agent for biocontrol of Croftonweed. Its production-toxin, a main pathogenic factor, has obvious bioactivity of weed control. However, the yield of toxin produced by A. alternata is lower. Consequently, it is no commercial prospects to produce the toxin by fermentation. In order to efficient use of the fungal secondary metabolite, it is necessary to study the biosynthesis metabolic process and toxin production related genes. In our lab an toxin-deficient mutant 001 have already been obtained by restriction enzyme-mediated integration(REMI). In present study, to elucidate the molecular mechanism of toxin biosynthesis of A. alternata, wild-type strain and toxin-deficient mutant strain 001 are used as experimental materials for physiological and biochemical comparisons. Based on the disrupted gene in the mutant 001 by plasmid pSH75, the disrupted gene HP001 and its downstream Gβ gene were cloned. Furthermore, it was also evaluated that the expression models of these two genes and the reason for resulting in the expression difference under different growth conditions. The interaction proteins of HP001 were identified through Yeast Two-hybrid method to reveal the gene function mechanism. To further probe the molecular mechanism of toxin biosynthesis, the expression difference genes between A. alternata wild-type strain and mutant strain were screened using the method of Suppression Subtractive Hybridization (SSH). This will provide the theoretical basis to improve toxin metabolic efficiency through genie recombination and to further develop A. alternata as a bioherbicide. The main study results are given as followings:1. The cloning of disrupted gene of A. alternata mutant 001Based on the known inserted sequence in plasmid pSH75, the mutant partial disrupted gene sequence with a 485bp fragment after alignment was isolated by using anchored PCR method. It was found that the inserted site was located in the multiple cloning sites, in which the plasmid sequence excluded. Hence, this indicated the fragment was the partial sequence which was disrupted by plasmid pSH75 in A. alternata. The full length of DNA and cDNA sequences for the HP001 gene were cloned using anchored PCR and RACE from A. alternata wild-type strain. The full length of DNA sequence is 2775bp, and contained one intron and two extrons. The full length of cDNA sequence is 1326bp, and encoded 442 amino acids.84% identities were acquired when compared to repress acid phosphatase of Pyrenophora tritici-repentis Pt-1C-BFP. However, the ratio of identity to histidine acid phosphatase family is maxium. Its molecular weight is 49.1 Kda. Although HP001 gene has not RHG motif of histidine acid phosphatase, it has the characteristically conserved domain and function activation region of this family members. So it is proposed that this gene may belong to histidine acid phosphatase family.HP001 gene fragment was used as probe to detect the gene copy in wild-type strain and disruption situation in mutant 001. Evidence from Southern blot experiments showed that HP001 was a single copy gene, moreover, the fragment of this gene in mutant 001 was larger than in wild-type, which indicated that HP001 gene was truly inserted by plasmid, resulting in an increase of the fragment. And the results of hygromycin gene as probe also showed that pSH75 was simply inserted to HP001 gene, and the insertion site just located at its 1130bp of full DNA sequence.2. Comparison of the difference in mechanism of toxin production and metabolism between A. alternata wild-type strain and mutant 001Through comparison of growth characters between A. alternata wild-type and mutant 001, it was not found that there was significant difference in the growth rate on PDA medium between them. Both wild and mutant had similar responses to different media. This indicated that HP001 gene was not a dependent growth factor. However, the sporulation in mutant was remarkedly reduced by 70%, which indicates that this gene related to sporulation. In PSK medium, a delayed phenomenon in aging black appeared in mutant 001, which indicated that the disruption of the gene led to a decline of utilization ratio of carbon source. Wild-type caused obvious disease spots and mutant did not on the leaf after plugs with mycelium were inoculated onto leaves for three days. It indicated that HP001 could regulate and control toxin production and pathogenic capacity of A. alternata. The spores of wild-type could germinate on onion epidermis or Croftonweed leaves, but the spores of mutant 001 not well on Croftonweed leaves. Such results showed that HP001 disruption didn’t affect spores germation, but could prevent the invasion, leading to lost pathogenicity. In low Pi concentration, wild-type strain could not normally grow, but mutant could grow well. The phosphatase activity in mutant 001 medium was significantly higher than that in wild-type in low-Pi. It showed that it activated some Pi-repressive gene which coded a secreted acid phosphatase after disruption HP001.The HP001 gene expression difference was compared to reveal HP001 gene function in submerged-culture or on leaves. The results showed that the gene expression appeared ascend tendency in submerged-cultrue on PSK medium, reached top at the sixth day and then fell. The early studies on the dynamics of toxin-production demonstrated similar results at the level of toxin production, which suggested that the expression of HP001 had a linear relationship to toxin production. In order to further study the trait of gene expression, wild-type strain were cultured in four different conditions, such as Croftonweed leaf epidermis, mesophyll tissues, and filter paper on petri dish with water or Croftonweed juice. The results showed that under the later two conditions, the HP001 gene expression kept much lower level and almost did not change. However, the gene expressed obviously on the leaves and reached top at 10h. The gene express level on the epidermis was higher than in mesophyll tissues. With mycelial plugs inoculated on wounded leaves, the gene expression performed in similar dynamics and rose to the level top at 10h. The expression level was higher than that cultured at 24h in PSK medium. Then, the gene expression level sharply declined and maitained stable low between 18h and 48h, and then just increased a little at 60h. This indicated that HP001 gene was specifically expressed during initial stage of infection as well as was closely related to the pathogenicity of A. alternata.RACE method was also used to clone the Gβ gene cDNA sequence, the result showed that the full coding sequence is 1056bp, encodes 352 amino acids, which has 88% homology with Setosphaeria turcica Gβ gene. The comparion results of the expression level between A. alternata wild-type and mutant 001, respectively showed that the Ga and Gβ genes had different expression level. However, the expression curve had not rethemic tendency. In the first two days, the expression levels in wild-type was lower than that in mutant, then suddenly increased at third day, then decreased, and increased again at fifth day. Overally, Ga and Gβ genes expression levels in wild-type always were higher than that in mutant during the later three days, however, and the expression tendency was nearly the same. But the decrease of Gβ in the expression level was more than Ga. The mecelia inoculated on leaves, the expression level of Ga and Gβ genes in mutant 001 was much lower than that in wild-type. Moreover, the decline tendency of Gβ genes in mutant 001 was more significantly as well. This indicated that the disruption of HP001 gene affected on G protein signal pathway, among which the influence on Gβ was stronger than on Ga.3. Study of interaction proteins of A. alternata HP001 geneBy constructing Yeast Two-Hybrid library of A. alternata, the library was screened by using HP001 gene as bait protein, and finally 79 blue colones were got. After blasting the sequencing results,3 kinds of proteins were found through making out the open reading frames (ORFs) which fused to GAL4AD sequence. The analysis of the sequences showed that the ORFs encoded 94,79 and 131 amino acids, respectively. These three proteins, 6-phosphogluconate dehydrogenase (6PGDH), glyceraldehydes-3-phosphate dehydrogenase (GAPDH) and glycosyl hydrolase (GLHY), had higher homology with Pyrenophora tritici-repentis Pt-1C-BFP, which were 95%,94% and 94% respectively. 6PGDH is the member of pentose phosphate pathway, GAPDH is mainly form protein in sugar decompose pathway, and GLHY belongs to cell wall degradation enzymes and pectin degradation enzymes. They are all different metabolism members, which may be possible virulence factors related to toxin production and invasion.4. Study of different expression genes between wild-type and mutant 001 of A. alternataA. alternata repression subtractive hybridization library were constructed, including the forward subtracted library using mutant as driver and wild-type as tester, and the reverse subtracted library using wild-type as driver, and mutant as tester. The results showed that 27 blots were found with significant difference in forward subtracted library, and 26 blots in reverse subtracted library. Forward subtracted library included Phosphoglycerate kinase, Cell division protease, Glyoxal oxidase and Trehalose phosphorylase etc. Reverse subtracted library included alcohol dehydrogenase, cytochrome oxidase subunit 2 (CoxⅡ), flavohemoprotein and so on. Besides, there are also some putative proteins and unknown genes in both libraries. Finally, only several paires of the genes screened from forward subtracted library were selected randomly to be further verified. Results showed that F28 (unknown gene), F75 (ribosomal protein) and F110 (glyoxal oxidase) were specifically expressed in wild-type strain, but not in mutant 001. |
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