| Morchella importuna is an edible and medicinal fungus with important economic value.On the one hand,many basic genetic problems in Morchella have not yet been revealed.It is urgent to invent an effective genetic technology to study the function of related genes.On the other hand,M.importuna is an edible fungus that requires soil cover cultivation,while the exchange of nutrients,including trace elements,between the mushroom and the soil is still obscure.Soil pollution,especially cadmium contamination,is a serious problem closely related to ecological environment,food safety and human health.With the expansion of the cultivated field,there is an increasing risk of heavy metal cadmium contamination in M.importuna.This study attempts to find the key gene for cadmium stress and to explain its function in cadmium metabolism.Copper chaperone protein(ATX1p)is involved in the transport of intracellular copper ions.Previous studies in yeast have shown that cadmium can bind with ATX1 p for cytotoxicity.ATX1 gene may play a key role in response to cadmium stress.To establish the genetic transformation system of M.importuna,we constructed the vector p1391-u-gus using the vector pCAMBIA1391 as a backbone,and cloned the Miubiquitin promoter of M.importuna to control the screening gene(Hyg)for hygromycin resistance and to control β-glucuronidase gene(GUS)as the one reporter gene;We cloned the glyceraldehyde-3-phosphate dehydrogenase gene(Migpd)promoter of M.importuna to control green fluorescent protein gene(eGFP)as another reporter gene.Agrobacteriummediated transformation method was used to transform the vector to the homokaryon of M.importuna(A50),and a total of 22 transformants were obtained.The conversion rate for the first round of screening was 8%.The results of PCR detection,green fluorescence observation and GUS staining showed that Agrobacterium-mediated transformation was successfully performed in M.importuna.In the research of genes for cadmium stress in M.importuna,the cadmium resistance of M.importuna was investigated by different concentrations of cadmium exogenous addition experiments under laboratory conditions,and the result showed M.importuna was sensitive to cadmium.The expression levels of candidate genes in M.importuna were determined under different cadmium concentrations.It was found that the expression of MiATX1 gene was down-regulated significantly,then the function of MiATX1 gene was studied by gene silencing and overexpression.MiATX1 silencing vector p1391-MiATX1-i and overexpression vector p1391-MiATX1-o were constructed,and Agrobacteriummediated transformation method was used to transform the two vectors to A50.As a result,four MiATX1 silencing transformants and four MiATX1 overexpressing transformants were obtained.Furthermore,the phenotype of the transformants was verified,and it was found that the MiATX1 gene silencing transformants showed enhanced cadmium resistance,while the MiATX1 gene overexpressing transformants showed reduced cadmium resistance.It indicates that the MiATX1 gene expression is negatively correlated with cadmium resistance of M.importuna.The above results indicated that the expression of exogenous genes and the verification of gene function were realized by genetic techniques such as gene transformation,gene silencing and gene overexpression for the first time in M.importuna.It is of great significance for the molecular genetic study of Morchella.In addition,the MiATX1 gene performed an important function on the cadmium resistance of M.importuna.This study is of great scientific and practical significance for profoundly revealing the molecular mechanism of cadmium metabolism of M.importuna and carrying out the breeding of new cadmium-resistant varieties of M.importuna. |
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