| Monascus spp.,are important medicinal and edible microorganisms in China,which were first screened from red mold rice(RMR)and characterized by van Tieghem(1884).The traditional fermentation food—Hongqu,which is produced by inoculating Monascus strains to steamed rice,has been used as food colorants,fermentation starters,and folk medicines for nearly two thousand years in oriental countries.Monasucs can conduct both sexual reproductions of forming ascospores and asexual reproductions of forming conidiospores in their life cycle.Conidia are the very important propagule of Monasucs.However,the yield of Monascus conidia is low,which makes them contaminated by other microorganisms in their growth process.This also largely affects the application of Monascus.At present,the genetic mechanisms of controlling the conidiophore formation had been addressed in Aspergillus nidulans as the model strains.Its conidiophore formation,contains five process:conidiophore stalk,vesicles,metulae,phialides and mature conidia,which is controlled by the central pathway of brl A-abaA-wet A.On the contrary,the conidiophore formation mechanism of Monascus is not clear.The results of genome analysis and micromorphological observations showed that no homolog hits of abaA were searched in the genome of Monascus.The central regulatory pathway in Monascus is brl A-wet A.The conidiophore formation in Monascus only contains formation process of conidiophore,vesicles and mature conidia.Their conidia are directly string in the top of the vesicles.Differentiation of metulae was not found in Monascus.In order to study the formation mechanism of the conidia,the abaA of Aspergillus nidulans was introduced into the genome of Monascus ruber M7 by heterologous expression technique.We gained 21 abaA expression strains.The phenotypic characters of colony and conidia were studied and analyzed.We also analyzed the yield and germination rate of conidiospores between M.ruber M7 and abaA expression strains.The main research results are as follows:1.The construction of abaA expression strains in M.ruber M7Based on the abaA gene of Aspergillus nidulans,the abaA expression cassette was constructed.Linking the expression cassette to the exspression vetor of p CAMBIA3300 with p MD19-T vector mediated,and then the gene expression vectors carrying abaA expression cassette were introduced into Monascus ruber M7 by Agrobacterium tumefaciens-mediated transformation.We gained 21 abaA expression strains by PCR and southern bloting.Nine strains whose codes were 1~#,4~#,5~#,6~#,9~#,16~#,18~#,21~#,22~#had one copy of abaA.Ten strains whose codes were 2~#,3~#,7~#,8~#,10~#,13~#,15~#,17~#,19~#,20~#had two copys of abaA.Two strains whose codes were 11~#,14~#had three copys of abaA.2.Comparison of Characteristics between M.ruber M7 and the abaA expression strains2.1 Analysis of colonial morphology,growth rate,conidial phenotypeThe colonial morphology of M.ruber M7 was compared with abaA expression strains.21 abaA expression strains had no obvious difference except 1~#,10~#,13~#strains.1~#,22~#,3~#,8~#,11~#,14~#strains were to be selected to study their growth gate of colony and conidial phenotype.The results showed that the growth of 1~#,22~#,3~#,11~#,14~#were fast more than that in M.ruber M7.The results of phenotype of conidia showed that the majority of the conidia of 1~#,22~#,3~#,8~#,11~#,14 were string on top of vesicles,only a small part of the conidia were born in two-way or three-way on the top of vesicles.2.2 Analysis of the yields and germination rate of conidiosporesThe conidiospores production of 1~#,22~#,3~#,8~#,11~#,14~#was increased by 3.6%,-7.5%,51.8%,135.8%,277.2%,489.6%.With the increasing of abaA copy number,the yields of conidia were significantly increased.The results showed that the germination rates of conidiospores of M.ruber M7,22~#,3~#,11~#were 23.24±2.57%,31.54±2.21%,23.86±1.70%,41.31±1.62%after four hours.The initial germination rates of the three expression strains were higher than that of Monascus ruber M7.After ten hours,their germination rate were 91.93±1.15%,93.83±1.31%,85.51±2.04%,97.45±0.80%.Except 11~#,the germination rates of other strains more than 90%.2.2 Analysis of color-valueThe yield of yellow pigments(380 nm),orange pigments(470 nm),red pigments(520nm)in M.ruber M7 was 1247.40±32.22 U/g,804.83±36.60 U/g,1080.03±36.42 U/g.Its production of pigments would reach the maximum after fifteen days.However,the pigmenst yield among 22~#,3~#,11~#would reach the maximum after twelve days.The pigments yield of22~#was 2141.87±120.71 U/g,803.37±79.47 U/g,1035.43±114.69 U/g.The pigment yield of 3~#was 2655.40±53.00 U/g,1419.20±355.10 U/g,1466.10±229.00 U/g,and the pigments yield of 11~#was 2046.70±120.70 U/g,807.60±48.90 U/g,1413.10±46.40 U/g.The extracellular pigments yield had no obvious change between abaA expression strains and M.ruber M7.3 Analysis of conidia proteomicsThe proteomics of conidiospores gained from abaA expression strains of 22~#,3~#,11~#and M.ruber M7 were analysed to investigate their differential expression proteins.Compared to the M.ruber M7,the number of up-regulated proteins about energy production and conversion,carbohydrate transport and metabolism in 22~#strain were more than down-regulated proteinswhile the number of up-regulated proteins about translation,ribosomal structure and biogenesis in 22~#strain were less than down-regulated proteins.Compared to the M.ruber M7,the number of up-regulated proteins about energy production and conversion,amino acid transport and metabolism in 3~#strain were more than down-regulated proteins while the number of up-regulated proteins about posttranslational modification,protein turnover,chaperones were less than down-regulated proteins.Compared to the M.ruber M7,the all proteins about secondary metabolites biosynthesis,transport and catabolism carbohydrate in 11~#strain were up-regulated.The number of up-regulated proteins about amino acid transport and metabolism were more than down-regulated proteins while the number of up-regulated proteins about energy production and conversion,translation,ribosomal structure and biogenesis were less than down-regulated proteins.The all proteins about RNA processing and modification,chromatin structure and dynamics were down-regulated. |
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