Scientia Agricultura Sinica ›› 2019, Vol. 52 ›› Issue (20): 3672-3684.doi: 10.3864/j.issn.0578-1752.2019.20.017

• FOOD SCIENCE AND ENGINEERING • Previous Articles     Next Articles

The Extracellular Enzymes Activity of F1 Generations and Single-spore Isolations in Agrocybe salicacola Strain YAAS711

HuiMing ZHOU1,2,YongChang ZHAO2(),HongMei CHAI2,YanZhen ZHANG1,2   

  1. 1 School of Biotechnology and Engineering, West Yunnan University, Lincang 677000, Yunnan
    2 Institute of Biotechnology and Germplasmic Resource, Yunnan Academy of Agricultural Sciences, Kunming 650205
  • Received:2019-04-08 Accepted:2019-08-03 Online:2019-10-16 Published:2019-10-28
  • Contact: YongChang ZHAO E-mail:yaasmushroom@aliyun.com

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Abstract:

【Objective】 The objective of this study was to explore the changes in activities of extracellular enzymes (EEA) of single-spore isolations and its self-crossing individuals from Agrocybe salicacola YAAS711 and to provide evidence for its genetic breeding. 【Method】 Eighteen single-spore monokaryons and 22 dikaryons from self-crossing progeny of A. salicacola YAAS711 were used as the test materials. The activities of CMCase, filter paper cellulase, β-glucanase, hemicellulase, amylase, protease, laccase, polyphenolase and peroxidase of all test strains were determined by 3,5-dinitrosalicylic acid (DNS), Folin methods and guaiacol method, and correlation analysis were carried out between the 9 EEs with mycelial growth rate, polarities and mating factors.【Results】 The results of 9 EEs showed that most of the both monokaryons and dikaryons had higher activities of amylase, filter paper cellulase, hemicellulose, β-glucanase, CMCase and protease, but their activities of peroxidase, polyphenolase and laccase were low. The mycelial growth rate of monokaryons and dikaryons had a certain degree of correlation with the average activity of 9 EEAs. Compared with the dikaryons derived from the combination of “fast-fast” growth rate of monokaryons, the dikaryons derived from the combination of “fast-slow” growth rates of monokaryons had higher EEAs, except hemicellulase, protease and polyphenol oxidase activities. However, there were no rules to follow, when it came to the combination of dikaryons from the cross of type II monokaryons. The indirect effect of polyphenolase on the mycelial growth rate of monokaryons was greater than that of laccase, but the combined effect of laccase on the mycelial growth rate of monokaryons was the largest. The synergistic effect among EEs had little effect on the mycelial growth rate of dikaryons, among them, laccase had the greatest direct effect on its mycelial growth rate. Multiple comparisons showed that the effect of polarity on EEs was greater than that of mating factors at a significant level of 0.05.【Conclusion】 In short, there was a significant correlation between the anomalies of the mycelial growth rate of the monokaryons from same polarity single spore isolations and the EEAs of F1 generations deriverd from these monokaryons in A. salicacola strain YAAS711. The EEAs of type I strains had same rule, but the differences were existed in some type II monokaryons and the dikaryons. There were certain correlation between the growth rate of monokaryons, EEAs and polarity, there were no direct correlation between mating factors and EEAs.

Key words: Agrocybe salicacola, self-crossing F1 generations, extracellular enzyme activity, monokaryon, dikaryon

Table 1

Test strains"

菌株Strain AxBx (F) AyBy (S) AxBy (F) AyBx (F)
单核体
Monokaryotic strain		 001、130、173、
185		 020、029、032、
071、187		 026、034、036、048、
076、163、182		 096、186
双核体
Dikaryotic strain		 F×F: 001*020、001*032
F×S: 001*029、001*071、001*187
S×S: 071*130、071*173、071*185
F×S: 020*130、020*173、020*185、
032*130、032*173、032*185		 F×F: 036*096、036*186、096*034
F×S: 096*026、096*048、096*076、
096*163、096*182

Fig. 1

Standard curve A: Glucose standard curve; B: Xylose standard curve; C: Tyrosine standard curve"

Table 2

The differences of the mycelial growth rate and extracellular enzyme average activities of different monokaryons from A. salicacola strainYAASM711"

单核体
Monokaryon		 菌丝生长速度
Mycelial growth rate (cm?d-1)		 胞外酶平均活力 Average activity of extracellular enzymes (U)
CMC FP βG HC AM PR LA PPO POD
001 0.30±0.01c 3.20±0.68a 3.89±0.60bcdef 1.55±0.35cde 2.77±0.88bcdef 7.59±0.86a 1.48±0.33abcd 0.77±0.04b 0.27±0.06fg 0.39±0.06a
020 0.39±0.01a 0.10±0.03f 3.43±0.41cdefgh 1.42±0.21cde 3.41±0.37ab 6.28±0.13b 1.21±0.03abcde 0.70±0.02c 0.58±0.03b 0.19±0.03d
026 0.15±0.00e 2.68±0.58ab 3.20±0.46defgh 2.11±0.58bcd 2.57±0.53cdefg 7.80±0.08a 1.50±0.55abc 0.13±0.01fgh 0.34±0.08ef 0.31±0.05ab
029 0.14±0.02e 1.28±0.19de 5.23±1.28ab 1.73±0.23cde 3.04±0.28abcde 2.02±0.26e 0.89±0.11cde 0.07±0.01h 0.10±0.01jk 0.35±0.07a
032 0.34±0.03b 1.92±0.90bcd 4.67±0.12bcd 3.17±1.77ab 3.00±0.38abcde 5.47±0.19c 1.22±0.11abcde 0.69±0.02c 0.61±0.07ab 0.21±0.05cd
034 0.23±0.01d 2.34±0.57abc 2.74±0.67fgh 1.75±0.15cd 2.59±0.47cdefg 4.21±0.43d 1.22±0.18abcde 0.18±0.07ef 0.18±0.05hi 0.36±0.04a
036 0.33±0.01b 2.30±0.56abc 4.42±0.85bcde 3.09±0.73ab 2.40±0.05defg 4.27±0.30d 1.27±0.18abcde 0.06±0.01h 0.42±0.08d 0.31±0.02ab
048 0.15±0.01e 0.15±0.04f 3.54±1.41cdefg 1.48±0.22cde 3.13±0.14abcd 0.95±0.40f 0.81±0.01e 0.15±0.02efg 0.21±0.04ghi 0.35±0.03a
071 0.14±0.01e 2.29±0.12abc 4.75±0.82bc 0.42±0.19e 1.89±0.20g 3.66±0.73d 1.34±0.32abcde 0.11±0.01fgh 0.15±0.02ij 0.35±0.08a
076 0.11±0.01f 0.66±0.56ef 6.18±0.85a 1.35±0.84cde 2.24±0.16efg 1.22±0.50f 0.81±0.04e 0.08±0.01gh 0.23±0.02gh 0.32±0.03ab
096 0.33±0.01b 2.56±0.41ab 1.99±0.83h 3.46±0.86a 2.59±0.69cdefg 5.36±0.12c 1.73±0.85ab 0.87±0.02a 0.31±0.04f 0.36±0.02a
130 0.15±0.01e 1.15±0.56de 3.85±0.44bcdef 1.28±0.52de 3.04±0.20abcde 2.11±0.40e 0.84±0.05de 0.39±0.08d 0.04±0.01k 0.31±0.06ab
163 0.15±0.00e 2.27±0.33abc 2.36±0.97gh 1.65±0.77cde 3.21±0.74abc 0.83±0.35f 0.90±0.07cde 0.11±0.09fgh 0.14±0.03ij 0.34±0.06a
173 0.15±0.00e 2.42±0.20abc 3.16±0.50efgh 1.57±0.95cde 3.62±0.32a 7.66±0.54a 1.16±0.43abcde 0.15±0.01efg 0.50±0.03c 0.30±0.08abc
182 0.13±0.01e 2.64±0.25abc 4.78±0.74bc 2.61±0.37abc 3.66±0.16a 5.12±0.58c 1.13±0.24bcde 0.15±0.01efg 0.40±0.02de 0.34±0.01a
185 0.15±0.00e 0.64±0.07ef 3.46±0.17cdefg 0.91±0.55de 2.02±0.35fg 0.90±0.43f 0.94±0.01cde 0.22±0.07e 0.27±0.03fg 0.31±0.06ab
186 0.35±0.03b 1.53±0.58cde 4.59±0.16bcde 1.52±0.21cde 2.41±0.13cdefg 4.37±0.11d 1.79±0.61a 0.65±0.02c 0.66±0.03a 0.34±0.08a
187 0.15±0.00e 1.23±0.61de 3.54±0.94cdefg 1.45±0.36cde 3.04±0.07abcde 1.96±0.51e 0.88±0.11cde 0.10±0.02gh 0.23±0.01gh 0.32±0.03bcd

Table 3

The differences of the mycelial growth rate and extracellular enzyme average activities of different dikaryons from A. salicacola strainYAASM711"

双核体
Dikaryon		 菌丝生长速度
Mycelial growth rate (cm?d-1)		 胞外酶平均活力 Average activity of extracellular enzymes (U)
CMC FP βG HC AM PR LA PPO POD
001*020 0.29±0.01g 2.96±0.22bcde 5.13±0.68bcde 3.07±0.22ab 1.61±0.51de 6.00±0.72cdef 1.33±0.26abcd 0.87±0.02b 0.47±0.03cd 0.28±0.02def
001*029 0.32±0.02def 3.53±0.10abcd 4.30±1.21cdefg 3.22±0.93ab 3.31±0.72a 10.09±0.97a 1.18±0.21abcd 1.26±0.09a 0.26±0.02gh 0.48±0.04a
001*032 0.30±0.01fg 0.88±0.64h 4.87±0.20cdef 3.13±0.25ab 3.09±0.40a 6.83±0.31bcd 1.50±0.05abcd 0.74±0.22b 0.42±0.07cde 0.27±0.10def
001*071 0.30±0.03fg 3.85±0.26abc 5.02±0.20bcdef 3.18±0.88ab 3.29±0.55a 6.77±0.40bcd 1.03±0.67bcd 1.36±0.43a 0.23±0.05hi 0.45±0.10ab
001*187 0.35±0.00abc 3.52±0.44abcd 4.25±0.52cdefg 3.23±0.72ab 3.28±0.50a 6.90±0.84bc 1.29±0.30abcd 1.30±0.03a 0.20±0.04hi 0.34±0.03cde
020*130 0.34±0.00bcd 4.54±0.79a 3.44±0.75efgh 1.58±0.42def 1.35±0.36ef 7.85±0.27b 1.62±0.28abcd 1.13±0.03a 0.35±0.11efg 0.27±0.10def
020*173 0.36±0.02ab 2.96±0.51bcde 4.52±0.59cdefg 2.70±0.51bcd 2.95±0.34abc 9.38±0.20a 1.48±0.40abcd 1.22±0.18a 0.27±0.04gh 0.25±0.02defg
020*185 0.33±0.01bcde 4.56±0.66a 6.60±1.24b 3.19±0.60ab 0.45±0.09f 4.98±0.49fgh 1.28±0.31abcd 1.13±0.01a 0.49±0.11c 0.18±0.03fgh
032*130 0.37±0.02a 3.50±0.70abcd 5.98±0.51bc 3.02±0.61abc 1.87±0.59bcde 7.00±0.16bc 1.64±0.93abc 1.23±0.04a 0.28±0.05fgh 0.23±0.04efgh
032*173 0.35±0.01abc 3.00±0.92bcde 4.75±0.44cdefg 3.40±0.87ab 0.88±0.24ef 7.00±0.92bc 1.18±0.36abcd 1.27±0.19a 0.35±0.02efg 0.14±0.01h
032*185 0.35±0.01abc 4.06±0.12ab 2.44±0.93h 1.78±0.16cdef 1.87±0.50cde 5.56±0.55defg 1.52±0.10abcd 1.23±0.02a 0.19±0.02hi 0.24±0.03defgh
071*130 0.22±0.00h 2.63±0.52cde 4.43±0.33cdefg 0.78±0.77f 1.56±0.28de 3.42±0.10i 0.95±0.03cd 0.10±0.01c 0.67±0.04ab 0.32±0.06cde
071*173 0.21±0.01hi 1.49±0.46gh 3.42±0.65efgh 1.43±0.42ef 2.68±0.91abcd 1.23±0.34j 0.86±0.05d 0.06±0.02c 0.59±0.03b 0.35±0.03bcd
071*185 0.21±0.01hi 2.09±0.81ef 4.64±0.84cdefg 0.74±0.09f 1.94±0.25bcde 7.35±0.48bc 0.99±0.17cd 0.09±0.02c 0.74±0.07a 0.35±0.07bcd
036*096 0.23±0.01h 2.61±0.73de 3.96±0.73defgh 2.90±0.39abc 1.99±0.91bcde 4.18±0.53hi 1.89±0.73a 0.70±0.01b 0.38±0.05def 0.16±0.03gh
036*186 0.19±0.01i 2.71±0.30cde 3.33±0.61fgh 2.89±0.87abc 1.52±0.47e 4.63±0.63ghi 1.79±0.10ab 0.71±0.04b 0.38±0.11def 0.28±0.01def
096*026 0.31±0.01efg 3.21±0.71bcde 4.31±0.51cdefg 1.13±0.72ef 1.91±0.96bcde 6.19±0.65cdef 1.16±0.37abcd 1.20±0.06a 0.35±0.04efg 0.14±0.10h
096*034 0.29±0.02g 3.14±1.05bcde 3.04±0.66gh 2.82±0.90abc 3.57±0.08a 4.68±0.56gh 1.70±0.09abc 1.34±0.27a 0.38±0.02def 0.25±0.01defg
096*048 0.30±0.01fg 2.91±0.68bcde 4.64±1.01cdefg 2.30±0.96bcde 2.98±0.67ab 7.15±1.34bc 1.29±0.30abcd 1.21±0.19a 0.22±0.03hi 0.41±0.01abc
096*076 0.31±0.01fg 3.48±0.85abcd 4.19±2.25defg 4.05±0.52a 1.93±0.22bcde 7.26±1.53bc 1.47±0.11abcd 1.22±0.01a 0.14±0.02i 0.31±0.10cde
096*163 0.33±0.03cde 2.84±0.22bcde 8.30±1.48a 3.17±0.44ab 1.99±0.82bcde 6.42±0.79cde 1.35±0.37abcd 1.22±0.08a 0.25±0.09gh 0.41±0.05abc
096*182 0.29±0.00g 3.44±0.70abcd 5.36±0.70bcd 2.86±0.88abc 3.14±0.94a 5.37±0.62efgh 1.39±0.57abcd 1.21±0.06a 0.29±0.02fgh 0.24±0.01defgh

"

项目 Item CMC FP βG HC AM PR LA PPO POD
CMC 1.000
FP -0.226 1.000
βG 0.408 -0.149 1.000
HC -0.007 -0.205 0.224 1.000
AM 0.590** -0.183 0.358 0.220 1.000
PR 0.558* -0.214 0.360 -0.268 0.700** 1.000
LA 0.090 -0.225 0.316 0.043 0.453 0.609** 1.000
PPO 0.038 0.072 0.383 0.179 0.575* 0.531* 0.467 1.000
POD 0.408 -0.080 -0.144 -0.272 -0.197 0.112 -0.225 -0.570* 1.000
菌丝生长速度
Mycelial growth rate (cm?d-1)		 0.089 -0.161 0.469* -0.033 0.473* 0.644** 0.785** 0.664** -0.370

Table 5

Path analysis by using mycelial growth rate as dependent variable"

自变量
Independent variable		 相关系数
Related
coefficient		 直接通径系数
Direct path coefficient		 间接通径系数 Indirect path coefficient 决策系数（R2）
Decision coefficient
→LA（X7） →PO（X8） 间接通径系数总和
Total of Indirect path coefficient
LA（X7） 0.785 0.607 0.178 0.178 0.585
PPO（X8） 0.664 0.380 0.284 0.284 0.360

Table 6

Correlation coefficients among mycelial growth rate and average activity of extracellular enzymes"

项目 Item CMC FP βG HC AM PR LA PPO POD
CMC 1.000
FP 0.038 1.000
βG 0.189 0.327 1.000
HC -0.256 -0.188 0.135 1.000
AM 0.296 0.172 0.307 0.186 1.000
PR 0.173 -0.176 0.399 -0.041 0.090 1.000
LA 0.649** 0.169 0.612** 0.192 0.549** 0.358 1.000
PPO -0.470* -0.027 -0.617** -0.316 -0.475* -0.406 -0.864** 1.000
POD -0.092 0.129 0.030 0.508* 0.241 -0.407 -0.069 -0.106 1.000
菌丝生长速度
Mycelial growth rate (cm?d-1)		 0.554** 0.276 0.416 0.023 0.612** 0.158 0.814** -0.666** -0.134

Table 7

Effects of different polarity on the average activity of extracellular enzymes"

极性 Polarities 胞外酶平均活力 The average activity of extracellular enzymes (U)
CMC FP βG HC AM PR LA PPO POD
AxBx 1.85±0.38a 3.59±0.68a 1.32±0.59b 2.86±0.44a 4.56±0.56ab 1.10±0.21a 0.38±0.15b 0.27±0.03b 0.33±0.07a
AyBy 1.36±0.39a 4.32±0.71a 1.64±0.55ab 2.87±0.26a 3.88±0.36bc 1.11±0.14a 0.33±0.02b 0.33±0.03b 0.28±0.05a
AxBy 1.86±0.50a 3.89±0.85a 2.00±0.52ab 2.83±0.32a 3.49±0.38c 1.09±0.18a 0.12±0.05c 0.27±0.05b 0.28±0.03a
AyBx 2.04±0.50a 3.29±0.50a 2.49±0.54a 2.50±0.41a 4.87±0.12a 1.76±0.73a 0.76±0.02a 0.49±0.04a 0.35±0.05a

Table 8

Effects of different mating factors on the average activity of extracellular enzymes"

交配因子 Mating factor 胞外酶平均活力 The average activity of extracellular enzyme (U)
CM FP βG HC AM PR LA PO PE
Ax 1.86±0.45a 3.78±0.79a 1.76±0.55a 2.84±0.36a 3.88±0.44a 1.09±0.19a 0.22±0.08b 0.27±0.04c 0.67±0.15a
Ay 1.56±0.42a 4.03±0.65a 1.88±0.55a 2.77±0.30a 4.16±0.29a 1.30±0.31a 0.45±0.02a 0.38±0.03a 0.30±0.05b
Bx 1.92±0.42a 3.49±0.62a 1.71±0.57a 2.74±0.43a 4.66±0.41a 1.32±0.38a 0.51±0.11a 0.34±0.03ab 0.34±0.06b
By 1.65±0.45a 4.07±0.79a 1.85±0.54a 2.85±0.30a 3.65±0.37a 1.10±0.16a 0.21±0.03b 0.30±0.04bc 0.31±0.04b
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