新疆野生阿魏菇原生质体的再生与单核菌株交配型测定

doi:10.6048/j.issn.1001-4330.2022.05.025

新疆农业科学 ›› 2022, Vol. 59 ›› Issue (5): 1245-1251.DOI: 10.6048/j.issn.1001-4330.2022.05.025

• 植物保护·微生物·贮藏保鲜加工 • 上一篇下一篇

新疆野生阿魏菇原生质体的再生与单核菌株交配型测定

努尔孜亚·亚力买买提¹(), 罗影¹, 郝敬喆¹, 祁颢萱³, 裴龙英², 孙春花⁴, 贾文捷¹(), 贾培松¹()

1.新疆农业科学院植物保护研究所/农业部西北荒漠绿洲作物有害生物综合治理重点实验室,乌鲁木齐 830091
2.新疆农业大学,乌鲁木齐 830091
3.新疆理工学院,新疆阿克苏 843000
4.青河县农业技术推广中心,新疆阿勒泰 836200

收稿日期:2021-12-05 出版日期:2022-05-20 发布日期:2022-06-09
通信作者: 贾文捷,贾培松
作者简介:努尔孜亚·亚力买买提(1977-),女,新疆人,副研究员,研究方向为食用菌资源, (E-mail) nrzy729@163.com
基金资助:
自治区自然科学基金计划少数民族特培项目(2019D03020);新疆农业科学院青年科技骨干创新能力培养项目(xjnkq-2021017);自治区教育厅高校科研计划项目(XJEDU2020Y047)

Regeneration and Mating Type Determination of Protoplast of Pleurotus ferulae

Nurziya Yarmamat¹(), LUO Ying¹, HAO Jingzhe¹, QI Haoxuan³, PEI Longying², SUN Chunhua⁴, JIA Wenjie¹(), JIA Peisong¹()

1. Key Laboratory of Integrated Crop Pest Management in China North-western Desert Oasis,Ministry of Agriculture P. R. China / Research Institute of Plant Protection, Xinjiang Academy of Agricultural Sciences, Urumqi, 830091
2. Xinjiang Agricultural University, Xinjiang, Urumqi 830091
3. Xin jiang Institute of Technology,Akesu Xinjiang 843000
4. Qinghe County Agricultural Technology Extension Center,Altai Xinjiang 836200

Received:2021-12-05 Online:2022-05-20 Published:2022-06-09
Correspondence author: JIA Wenjie, JIA Peisong
Supported by:
Special Training Program of Natural Science Foundation of Xinjiang Autonomous Region(2019D03020);Innovation Ability Training Project for Young Sci-Tech Backbone Talents Sponsored by Xinjiang Academy of Agricultural Sciences(xjnkq-2021017);University Scientific Research Project of Education Department of Xinjiang Autonomous Region(XJEDU2020Y047)

摘要/Abstract

摘要：

【目的】利用原生质体单核化技术获得具有优良性状的阿魏菇(Pleurotus ferulae)新菌种,为新疆野生阿魏菇新品种的保育及产业化应用提供技术支撑。【方法】采用原生质体制备技术获得阿魏菇单核菌株并进行交配型测定。【结果】酶解3.5 h时,PL01、PL163与 PL176等3株野生阿魏菇亲本菌株的原生质体释放量最大,浓度最高达20.6×10⁵个/ mL,通过优化的原生质体单核化技术最终获得50个单核菌株,亲本菌株PL163、PL01、PL176原生质体单核化率分别为 25.68%、12.8%、10.8%,获得率较低。对其进行了交配型测定,每株亲本菌株得到2种交配型的单核菌株。其中,菌株PL01与PL163、 2种交配型获得比例均为1∶3,而PL1762种交配型获得比例为1∶4。每个亲本菌株与获得的单核菌株在菌丝长速、菌落形态方面均具有差异。以不同交配型的单核菌株PL01-P16、PL01-P49、PL163-7、PL163-24 、PL176-23、PL176-39作为杂交育种材料进行配对,获得12株性状优良的杂交菌株。拮抗反应表明,12株杂交菌株与亲本之间形成明显隆起的拮抗带,与亲本菌株显著不同。【结论】3株阿魏菇野生菌株在原生质体再生单核获得率、长速、形态等方面具有丰富的多样性,获得的杂交菌株为新疆阿魏菇新品种的保护奠定了基础,并为选育优良阿魏菇杂交品种提供了良好的种质材料。

关键词: 野生阿魏菇(P. ferulae); 原生质体单核化; 杂交育种; 交配型测定

Abstract:

【Objective】 To obtain a new strain of Pleurotus ferulae with excellent traits by the protoplast monokaryotation technology in the hope of laying the theoretical and technical foundation for the selection and industrial production of new varieties of wild P. ferulae in Xinjiang. 【Method】 The protoplasmic preparation technique was used to obtain the mononuclear strain of P. ferulae and determine its mating type. 【Result】 At 3.5 h after enzymolysis, the protoplast release of the 3 wild P. ferulaeparent strains PL01, PL163 and PL176 was the maximum, with the concentration as high as 20.6 × 10⁵ /mL. Through optimized protoplast monokaryonization, 50 mononuclear strains were finally obtained from the 3 parent strains. The protoplast monokaryonization rates of parent strains PL163, PL01 and PL176 were 25.68%, 12.8% and 10.8%, respectively, with a low acquisition rate. Then, the mating type was determined, and mononuclear strains with 2 mating types were obtained from each parent strain. Among them, the acquisition ratio of the 2 mating types from PL01 and PL163 strains was 1∶3, while that of PL176 was 1∶4. Differences were found in mycelial growth rate and colony morphology among each parent strain and the obtained mononuclear strains. Subsequently, pairing was performed using mononuclear strains PL01-P16, PL001-P49, PL163-7, PL163-24, PL176-23 and PL176-39 with different mating types as hybrid breeding materials, and 12 hybrid strains with excellent traits were obtained. The antagonistic reaction showed that the 12 hybrid strains formed an obvious uplifted antagonistic band between their parents, and were significantly different from their parents. 【Conclusion】 The 3 wild P. ferulae strains present rich diversity in protoplast regeneration, mononuclear acquisition rate, growth rate and morphology. The obtained hybrid strains lay a foundation for the conservation of new varieties of P.

Key words: Pleurotus ferulae; protoplasts mononucleate; hybridization; mating type determination

中图分类号:

S188

努尔孜亚·亚力买买提, 罗影, 郝敬喆, 祁颢萱, 裴龙英, 孙春花, 贾文捷, 贾培松. 新疆野生阿魏菇原生质体的再生与单核菌株交配型测定[J]. 新疆农业科学, 2022, 59(5): 1245-1251.

Nurziya Yarmamat, LUO Ying, HAO Jingzhe, QI Haoxuan, PEI Longying, SUN Chunhua, JIA Wenjie, JIA Peisong. Regeneration and Mating Type Determination of Protoplast of Pleurotus ferulae[J]. Xinjiang Agricultural Sciences, 2022, 59(5): 1245-1251.

图/表 9

图1 阿魏菇菌株释放出原生质体

Fig.1 The protoplasts released by Pleurotus ferulae

表1 原生质体单核菌株的数量以及单核获得比例

Table1 The quantity of monocytic strains and the rate of monokaryogenesis

菌种 Strain	挑取数量 Picking number	萌发数量 Germination number	单核数量 Mononuclear number	单核获得率 Mononuclear Acquisition rate (%)
PL01	198	156	20	12.8
PL163	89	74	19	25.68
PL176	175	108	11	10.18

表2 交配型分类结果

Table 2 The classification of mating type

亲本菌株 Parent strain	不同交配型的阿魏菇单核菌株 Mononuclear strains of P. eryngii with different mating types
PL01	3、19、49、65、236
PL01	14、16、17、27、45、4、51、53、56、61、66、70、234、237、264
PL176	22、39
PL176	18、23、56、71、75、79、88、146、148
PL163	2、10、24、89
PL163	1、5、7、8、54、39、40、43、49、50、51、56、78、79、80

图2 原生质体产量随不同酶解时间的变化 (个 /mL)

Fig.2 The production of Ganoderma lucidum protoplast in different time(ind /mL)

表3 亲本菌株与单核菌株的生长速度

Table 3 The growth speed of mononuclear strains

菌株 Strain	菌丝长速 Growth rate (mm/d)	菌株 Strain	菌丝长速 Growth rate (mm/d)
PL01	5.28±0.08^a	PL163-P08	4.70±0.03^c
PL01-P3	3.57±0.1 $2 d e$	PL163-P10	2.94±0.09^g
PL01-P14	2.81±0.24^f	PL163-P24	5.41±0.08^b
PL01-P16	5.07±0.2 $0 a b c$	PL163-P39	3.42±0.16^f
PL01-P17	3.31±0.07^e	PL163-P40	3.16±0.1 $5 f g$
PL01-P19	4.74±0.17^c	PL163-P43	3.49±0.0 $5 f g$
PL01-P27	4.78±0.09^c	PL163-P49	3.17±0.11^h
PL01-P45	4.74±0.01^c	PL163-P50	2.02±0.04ⁱ
PL01-P48	4.74±0.27^c	PL163-P51	2.35±0.10^h
PL01-P49	5.26±0.1 $3 d e$	PL163-P54	4.87±0.11^c
PL01-P51	3.56±0.1 $5 d e$	PL163-P56	3.61±0.21^f
PL01-P53	3.48±0.10^e	PL163-P78	3.28±0.10^f
PL01-P56	5.03±0.1 $3 a b c$	PL163-P79	4.26±0.02^d
PL01-P61	4.94±0.3 $6 a b c$	PL163-P80	2.92±0.04^g
PL01-P65	4.67±0.05^c	PL176	5.99±0.19^a
PL01-P66	3.96±0.21^d	PL176-P18	4.15±0.0 $5 b c d$
PL01-P70	4.85±0.14^c	PL176-P22	4.48±0.0 $3 c d e$
PL01-P234	4.77±0.10^c	PL176-P23	5.73±0.0 $3 a b c$
PL01-P236	4.88±0.0 $9 b c$	PL176-P39	5.08±0.15^a
PL01-P237	4.66±0.03^c	PL176-P56	4.40±0.3 $3 b c d$
PL01-P264	3.89±0.34^d	PL176-P71	4.89±0.0 $6 b c d$
PL163	6.20±0.11^a	PL176-P75	3.34±0.3 $2 d e$
PL163-P01	4.13±0.3 $0 d e$	PL176-P79	2.96±0.0 $4 b c d$
PL163-P02	3.26±0.29^f	PL176-P88	4.86±0.0 $8 d e f$
PL163-P05	3.92±0.06^e	PL176-P146	4.92±0.2 $5 d e f$
PL163-P07	6.08±0.07^a	PL176-P148	3.93±0.2 $1 e f$

表3 亲本菌株与单核菌株的生长速度

Table 3 The growth speed of mononuclear strains

菌株 Strain	菌丝长速 Growth rate (mm/d)	菌株 Strain	菌丝长速 Growth rate (mm/d)
PL01	5.28±0.08^a	PL163-P08	4.70±0.03^c
PL01-P3	3.57±0.1 $2 d e$	PL163-P10	2.94±0.09^g
PL01-P14	2.81±0.24^f	PL163-P24	5.41±0.08^b
PL01-P16	5.07±0.2 $0 a b c$	PL163-P39	3.42±0.16^f
PL01-P17	3.31±0.07^e	PL163-P40	3.16±0.1 $5 f g$
PL01-P19	4.74±0.17^c	PL163-P43	3.49±0.0 $5 f g$
PL01-P27	4.78±0.09^c	PL163-P49	3.17±0.11^h
PL01-P45	4.74±0.01^c	PL163-P50	2.02±0.04ⁱ
PL01-P48	4.74±0.27^c	PL163-P51	2.35±0.10^h
PL01-P49	5.26±0.1 $3 d e$	PL163-P54	4.87±0.11^c
PL01-P51	3.56±0.1 $5 d e$	PL163-P56	3.61±0.21^f
PL01-P53	3.48±0.10^e	PL163-P78	3.28±0.10^f
PL01-P56	5.03±0.1 $3 a b c$	PL163-P79	4.26±0.02^d
PL01-P61	4.94±0.3 $6 a b c$	PL163-P80	2.92±0.04^g
PL01-P65	4.67±0.05^c	PL176	5.99±0.19^a
PL01-P66	3.96±0.21^d	PL176-P18	4.15±0.0 $5 b c d$
PL01-P70	4.85±0.14^c	PL176-P22	4.48±0.0 $3 c d e$
PL01-P234	4.77±0.10^c	PL176-P23	5.73±0.0 $3 a b c$
PL01-P236	4.88±0.0 $9 b c$	PL176-P39	5.08±0.15^a
PL01-P237	4.66±0.03^c	PL176-P56	4.40±0.3 $3 b c d$
PL01-P264	3.89±0.34^d	PL176-P71	4.89±0.0 $6 b c d$
PL163	6.20±0.11^a	PL176-P75	3.34±0.3 $2 d e$
PL163-P01	4.13±0.3 $0 d e$	PL176-P79	2.96±0.0 $4 b c d$
PL163-P02	3.26±0.29^f	PL176-P88	4.86±0.0 $8 d e f$
PL163-P05	3.92±0.06^e	PL176-P146	4.92±0.2 $5 d e f$
PL163-P07	6.08±0.07^a	PL176-P148	3.93±0.2 $1 e f$

图3 菌龄7 d的单核及双核菌丝的显微结构注注:A:单核菌丝;B:双核菌丝,可见锁状联合

Fig.3 Microstructure of monokaryontic and multinucleated mycelia of 7 d Note: A: Monokaryontic mycelia ; B: Multinucleated mycelia,clamp connection by the black arrows

图4 亲本菌株与单核菌株菌落形态

Fig.4 Colony morphology of parent strain and monocyte strain

表4 杂交菌株编号

Table 4 hybrid strain

杂交菌株 Hybrid strain	杂交菌株 Hybrid strain
PL176P23×PL01P16	PL01P49×PL163P7
PL176P39×PL01P16	PL01P49×PL163P24
PL176P23×PL01P49	PL163P24×PL176P23
PL176P39×PL01P49	PL163P24×PL176P39
PL01P16×PL163P7	PL163P7×PL176P23
PL01P16×PL163P24	PL163P7×PL176P39

图5 亲本菌株与杂交菌株的拮抗实验

Fig.5 Antagonism test between parental strain and hybrid strain

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新疆野生阿魏菇原生质体的再生与单核菌株交配型测定

Regeneration and Mating Type Determination of Protoplast of Pleurotus ferulae

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