| Fuzhuan tea is a type of multifunctional healthy tea,which obtained its unique character and health care function when there were “golden flower fungi” growing on it,producing a series of secondary metabolite and these products could then react with tea substrate,changing into beneficial components.Lae A,a global regulator involved in the regulation of secondary metabolism and morphogenesis.Currently,nobody has used Aspergillus cristatus to study Lae A gene and its regulatory mechanism of secondary metabolism.In this study,gene knock technique was used to analyze the function of Lae A gene,which has vast importance in controlling harmful or beneficial products in Aspergillus cristatus,and clarifying the health care functions of fuzhuan tea,and improve tea quality as well.Gene knockout vector was constructed with the full-length information of Lae A.By Agrobacterium tumefaciens-mediated transformation,Lae A gene of Aspergillus cristatus was knocked out from its genome,and ?Lae A mutants was obtained.Then southern blot has been used to prove the knockout cassette whether ?Lae A mutant to be a single copy.HPLC technique was used to analyze differential metabolites between ?Lae A mutant and wild type strain,and the phenotype of the ?Lae A mutant was observed in detail.Here are the main results:1)The gene knockout vector p DHt/sk-hyg-Lae AL-Lae AR was constructed based on the homologous recombination principle that the target gene was replaced by the maker gene.More than 50 mutants were obtained through Agrobacterium tumefaciens-mediated transformation method and only two were ?Lae A mutants(number 5 and 6),two were random insertion mutants of T-DNA.2)The Lae A sequence of Aspergillus was downloaded from the NCBI(National Center for Biotechnology Information)database,and then compared with the local genomic database of Aspergillus cristatus to obtain the full-length sequence information of Lae A.The full-length of Lae A c DNA is 1,183 bp,including a 67 bp untranslated regionat the 222 rd bp and a 1,116 bp open reading frame,presumably coding a protein with 371 amino acids,and the relative molecular mass and isoelectric point of this protein are 71.3k D and 5.97.Hydrophilic amino acids are rich in this protein,but no transmembrane spiral structure and signal peptide structure were founded according to software prediction.3)Morphology comparison between wild type strain and ?Lae A mutant:On the 3rd day,?Lae A mutant was a little bit whiter at the middle part and had less hypha than wild type strain.Meanwhile,less ascospores were found in ?Lae A mutant under stereomicroscope,which may be caused by the deficiency of sexual development.However,two days latter no obvious difference was discovered between them,and the dense of cleistothecium return to a same level.We did not see any apparent differences in both asexual and sexual structure of wild type strain and ?Lae A mutant at further observation under oil immersion lens.However,we found that there were much more conidia in Lae A knockout mutant comparing to wild type.4)High Performance Liquid Chromatography analysis:The content of several main products increased dramatically in ?Lae A mutant,and wild type strain had 4 extra compounds than knock out strain.All these results firmly proved that Lae A gene did involve in the regulation of secondary metabolism,and missing of Lae A gene could facilitate the expression of some gene clusters in Aspergillus cristatus,which increased the content of several specific dominant products. |
PDF Full Text Request