Study On Characters Of Some Mutants From Transformation Library Of Monascus M-7 Mediated By Agrobacterium Tumefaciens

In this study,8132 transformants from T-DNA insertion library of Monascus ruber mediated by Agrobacterium tumefaciens were used as materials.Some of them with significant changes were selected through the colony and micro-morphological observation,and classified into seven groups.On this basis,the main metabolites of the representatives of 14 mutants were analyzed.The research contents were as follow:1.The expansion of transformation library of Monascus M-7 mediated by Agrobacterium tumefaciensReferring to the Agrobacterium-mediated transformation condition of Monascus M-7 established by our lab before:original strain M-7 was cultivated on the WA medium for 20 d at 30℃,concentration of Monascus spores was 10~5/mL,optical density value at 600 nm of Agrobacterium was 0.5,concentration of inducer AS was 80 retool/L,Agrobacterium and Monascus spores were co-cultured for 3 d at 30℃.The number of transformants in the library was amplifid to 8 132.2.Selection and morphologic observation of of transformants8 132 transformants were cultivated on the PDA medium for 15 d at 30℃. According to the colony observation,230 colonies which were significantly different form M-7 on color,growth rate,aerial hypha and other aspects,were selected and divided into seven categories:(1) relatively large diameter,ring-shaped distribution;(2) relatively large diameter,light-colored,frizzled aerial hypha;(3) relatively large diameter,light-colored,Liusu aerial hypha;(4) relatively large diameter,deeper color, frizzled aerial hypha;(5) relatively small diameter,flat colony,frizzled aerial hypha;(6) relatively smaller diameter,central uplift,shorter aerial hypha;(7) middle diameter, fan-shaped division.The result of morphology observation of some representative strains selected form each category showed that,the conidia shape,surface attachment on the mycelium,the volume and shape of cleistothecia also changed a lot. 3.Stability study and PCR identification of transformants35 transformants choosed from 230 transformants were cultivated on PDA medium for 5 generations,then transferred to PDA plate which containing 50μg/mL hygromcin B with original strain M-7 as control.At the same time,14 representatives of the them were identified by PCR according to the gus gene.The results showed that 14 strains could grow on the resistant plate after 6 generation-culture with resistance stability of 88.57%.All of them has T-DNA insertion of genome,with good genetic stability.4.Analysise of amylase and protease main metabolites of partial transformantsThe activity of amylase and protease of RFR produced by 14 transformants were measured by DNS method and Folin method.Starch-PAGE was Combined With DNS method to determination different active component of amylase.The results were as follow.The activity of amylase of 13 mutants were higher than M-7;1067~# was the higest up to 3227.10 U/g and was 20 times that of M-7. Protease activity of most transformants were higher than M-7.Acid protease activity of 878~# was the higest,reaching 1543 U/g,nearly 2 times that of M-7;neutral protease activity of 1067~# was almost 4 times that of M-7,which was 3211 U/g;alkaline protease activity of 2225~# was the higest reaching 1773 U/g,nearly 4 times that of M-7. Starch-PAGE could effectively detect the active components of amylase,the results showed that the more the active component the higher the activity of amylase.5.Analysis of amylase and protease main metabolites of partial transformantsUV-VIS,TLC,HPLC,ELISA and other methods were used on the mensuration of pigment,MonacolinK,GABA and citrinin of RFR produced by 14 transformants.The results showed that,the capacity of transformants to producing metabolites have taken place in different degrees compared with the original strain M-7,the ability to produce pigment of all the 14 transformants were poorer than M-7;806~# and 2553~# were the poorest,and the pigment-value of them were less than tenth of M-7. Monacolin K output of 10 transformants was higher than M-7;3245~# was almost 10 times that of M-7,which amounts to 679.25μg/g.GABA content of 178~# was the highest almost 3 times that of M-7,reaching 1.455g/kg.Citrinin production of 1822~# was the highest reaching 60.01 ng/g(dry weight of RFR),nearly 30 times that of M-7; citrinin production of 178~# and 806~# was not detected under this experimental conditions.