雄性二倍体毛白杨再生体系的构建和遗传转化的研究

doi:10.12302/j.issn.1000-2006.202012029

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南京林业大学学报（自然科学版） ›› 2022, Vol. 46 ›› Issue (1): 187-196.doi: 10.12302/j.issn.1000-2006.202012029

雄性二倍体毛白杨再生体系的构建和遗传转化的研究

俞子承(), 凌聪, 陈赢男, 李淑娴, 尹佟明, 李小平^*()

南京林业大学林学院,南方现代林业协同创新中心,江苏南京 210037

收稿日期:2020-12-19 接受日期:2021-04-14 出版日期:2022-01-30 发布日期:2022-02-09
通讯作者: 李小平
基金资助:
江苏省自然科学基金面上项目(BK20161525);国家重点研发计划(2016YFD0600101)

Establishment of regeneration system and study on genetic transformation for male diploid Populus tomentosa

YU Zicheng(), LING Cong, CHEN Yingnan, LI Shuxian, YIN Tongming, LI Xiaoping^*()

Co-Innovation Center for the Sustainable Forestry in Southern China, College of Forestry,Nanjing Forestry University, Nanjing 210037, China

Received:2020-12-19 Accepted:2021-04-14 Online:2022-01-30 Published:2022-02-09
Contact: LI Xiaoping

摘要/Abstract

摘要：

【目的】建立雄性二倍体毛白杨再生体系,构建稳定的遗传转化方法,为进一步研究毛白杨基因功能提供试验平台。【方法】以雄性二倍体毛白杨的幼嫩茎段和叶片为外植体,1/2 MS为基本培养基,通过调整6-BA、IAA和TDZ激素浓度进行再生体系筛选;采用农杆菌EHA105介导叶盘法,控制预培养时间、菌液浓度、侵染时间、共培养时间和卡那霉素筛选浓度,进行遗传转化;以幼嫩叶片原生质体为受体细胞,PEG介导转化荧光标记基因EGFP,进行瞬时表达。【结果】雄性二倍体毛白杨再生过程包括继代、芽伸长和生根3个阶段,其培养基组分分别为1/2 MS+0.5 mg/L 6-BA+0.5 mg/L NAA+0.005 mg/L TDZ、1/2 MS+0.5 mg/L 6-BA+0.3 mg/L NAA和1/2 MS+0.3 mg/L NAA+0.5 mg/L IBA,该条件下生根率为96.7%,增殖系数为4.47。遗传转化过程包括预培养、农杆菌侵染、共培养、抗性筛选和生根5个阶段,其中预培养为12 h、农杆菌浓度OD₆₀₀为0.4、侵染时间为20 min、共培养时间为24 h、卡那霉素(30 mg/L)筛选45 d和抗性苗生根20 d。试验共获得86株抗性植株,其中14株分子鉴定结果为阳性。在40% PEG4000的介导下,EGFP基因瞬间转化效率为50%。【结论】雄性二倍体毛白杨再生周期短、遗传转化稳定,是杨树基础研究的理想材料。本研究拓宽了杨树遗传转化体系,为杨树分子辅助育种提供了新途径。

关键词: 二倍体毛白杨, 再生体系, 遗传转化, 瞬时表达

Abstract:

【Objective】 The regeneration system and the stable genetic transformation method of male diploid Populus tomentosa was established to provide an experimental platform for further research on gene function.【Method】 The infancy stem segments and leaves of male diploid P. tomentosa were selected as explants and the regeneration system was established by optimizing the concentrations of 6-BA, IAA and TDZ, based on 1/2 MS basic medium. Agrobacterium tumefaciens EHA105 was used for genetic transformation via the leaf disc transformation method; it was combined with pre-culture time, bacterial concentration, infection time, co-culture time, and the kanamycin screening concentration. The protoplasts of young leaves were used as receptor cells and transiently transformed with the fluorescent marker gene, EGFP, using the polyethylene glycol-mediated method.【Result】 The regeneration process of male diploid P. tomentosa involves three stages: subculture, bud elongation, and rooting. The medium components were: 1/2 MS + 0.5 mg/L 6-BA + 0.5 mg/L NAA + 0.005 mg/L TDZ 1/2 MS + 0.5 mg/L 6-BA + 0.3 mg/L NAA and 1/2 MS + 0.3 mg/L NAA + 0.5 mg/L IBA; the rooting percentage was 96.7% and the multiplication coefficient was 4.47. The process of genetic transformation involved five stages: pre-culture, agrobacterium infection, co-culture, resistance screening and rooting. The specific plan was pre-culture for 12 h, with an agrobacterium concentration of OD₆₀₀ as 0.4, infection-time of 20 min, co-culture time of 24 h, kanamycin (30 mg/L) screening for 45 d, and resistant seedlings rooting for 20 d. A total of 86 resistant plants were obtained in this study; among them molecular identification of 14 plants were stable. The efficiency of the EGFP gene transformation was 50% under the guidance of 40% PEG4000.【Conclusion】 The male diploid P. tomentosa was rapidly regenerated and exhibited good transformation stability; it was considered an ideal material for basic polar research. This study broadened the genetic transformation system of poplar and provided a new approach for the molecular assisted breeding of poplar.

Key words: diploid Populus tomentosa, regeneration system, genetic transformation, transient expression

中图分类号:

S792.117

俞子承,凌聪,陈赢男,等. 雄性二倍体毛白杨再生体系的构建和遗传转化的研究[J]. 南京林业大学学报（自然科学版）, 2022, 46(1): 187-196.

YU Zicheng, LING Cong, CHEN Yingnan, LI Shuxian, YIN Tongming, LI Xiaoping. Establishment of regeneration system and study on genetic transformation for male diploid Populus tomentosa[J].Journal of Nanjing Forestry University (Natural Science Edition), 2022, 46(1): 187-196.DOI: 10.12302/j.issn.1000-2006.202012029.

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处理 treatment	消毒时间 disinfection time		种植数 planting number	污染数 pollution number	污染率 pollution rate	褐化率 browning rate	死亡率 death rate	显著性分析 sig.
处理 treatment	70%乙醇/s 70% ethanol	次氯酸钠/min sodium hypochlorite	种植数 planting number	污染数 pollution number	污染率 pollution rate	褐化率 browning rate	死亡率 death rate	显著性分析 sig.
1	20	5	50	46	0.92	0.55	0.34	a
2	20	10	50	43	0.86	0.62	0.25	b
3	20	15	50	39	0.78	0.72	0.36	ac
4	30	5	50	22	0.44	0.54	0.37	ac
5	30	10	50	28	0.56	0.55	0.44	d
6	30	15	50	28	0.56	0.48	0.86	e
7	40	5	50	37	0.74	0.50	0.70	f
8	40	10	50	33	0.66	0.52	0.62	g
9	40	15	50	30	0.60	0.53	0.77	h

培养基 culture medium	顶芽生长情况 the growth rate of bud
培养基 culture medium	10 d	20 d
1/4 MS	生长速度与WPM相似	生长速度最慢
1/2 MS	生长速度最快	生长速度最快
MS	生长速度最慢	生长速度与WPM相似
WPM	生长速度与1/4 MS相似	生长速度与MS相似

处理 treatment	外源激素/ (mg·L^-1) exogenous hormone		平均生根率/% average rooting rate	显著性分析 sig.
处理 treatment	NAA	IBA	平均生根率/% average rooting rate	显著性分析 sig.
1	0.3	0.3	53.3	bc
2	0.3	0.5	96.7	a
3	0.3	0.7	70.0	b
4	0.5	0.3	56.7	b
5	0.5	0.5	66.7	b
6	0.5	0.7	63.3	b
7	0.7	0.3	36.7	c
8	0.7	0.5	16.7	d
9	0.7	0.7	3.3	d

处理 treatment	外源激素/ (mg·L^-1) exogenous hormone			平均增殖系数 average multiplication coefficient	显著性分析 sig.
处理 treatment	6-BA	NAA	TDZ	平均增殖系数 average multiplication coefficient	显著性分析 sig.
1	0.3	0.3	0.005	1.60	c
2	0.3	0.5	0.010	2.10	c
3	0.3	0.7	0.015	2.70	c
4	0.5	0.3	0.015	4.47	abc
5	0.5	0.5	0.005	6.63	a
6	0.5	0.7	0.010	3.00	ab
7	0.7	0.3	0.010	3.43	bc
8	0.7	0.5	0.015	4.10	abc
9	0.7	0.7	0.005	4.03	abc

雄性二倍体毛白杨再生体系的构建和遗传转化的研究

Establishment of regeneration system and study on genetic transformation for male diploid Populus tomentosa

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图/表 14

参考文献 30

相关文章 11

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处理 treatment	外源激素/(mg·L^-1) exogenous hormone		30 d芽平均生长长度/mm average growth length of bud in 30 d
处理 treatment	6-BA	NAA	30 d芽平均生长长度/mm average growth length of bud in 30 d
1	0.1	0.1	16.0
2	0.1	0.3	18.0
3	0.1	0.5	19.0
4	0.3	0.1	15.0
5	0.3	0.3	20.0
6	0.3	0.5	24.0
7	0.5	0.1	23.0
8	0.5	0.3	26.3
9	0.5	0.5	23.3

处理 treatment	时间/h time	接种数/ 个 plants number	产生抗性芽叶盘数/ 个resistant buds number	抗性芽获得率/ %resistant buds rate	显著性分析 sig.
预培养 pre-culture	0	100	7	7	a
	12	100	8	9	b
	24	100	2	2	c
	48	100	0	0	d
共培养 co-cultivation	8	50	0	0	a
	16	50	2	4	a
	24	50	3	6	b
	32	50	1	2	a
	40	50	0	0	a
	48	50	1	2	a
	56	50	0	0	a

菌液浓度 OD₆₀₀	侵染时间/ min infection time	叶盘数 plants number	产生抗性芽的叶盘数 resistant buds number	抗性芽获得率/% resistant buds rate	显著性分析 sig.
0.2	10	45	0	0	a
	20	48	1	2	ab
	30	40	2	5	b
0.4	10	48	4	8	c
	20	47	5	11	c
	30	48	2	4	b
0.6	10	48	0	0	a
	20	48	0	0	a
	30	48	0	0	a
0.8	10	44	0	0	a
	20	42	0	0	a
	30	48	0	0	a

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菌液浓度 OD₆₀₀	侵染时间/ min infection time	叶盘数 plants number	产生抗性芽的叶盘数 resistant buds number	抗性芽获得率/% resistant buds rate	显著性分析 sig.
0.2	10	45	0	0	a
	20	48	1	2	ab
	30	40	2	5	b
0.4	10	48	4	8	c
	20	47	5	11	c
	30	48	2	4	b
0.6	10	48	0	0	a
	20	48	0	0	a
	30	48	0	0	a
0.8	10	44	0	0	a
	20	42	0	0	a
	30	48	0	0	a

菌液浓度 OD₆₀₀	侵染时间/ min infection time	叶盘数 plants number	产生抗性芽的叶盘数 resistant buds number	抗性芽获得率/% resistant buds rate	显著性分析 sig.
0.2	10	45	0	0	a
	20	48	1	2	ab
	30	40	2	5	b
0.4	10	48	4	8	c
	20	47	5	11	c
	30	48	2	4	b
0.6	10	48	0	0	a
	20	48	0	0	a
	30	48	0	0	a
0.8	10	44	0	0	a
	20	42	0	0	a
	30	48	0	0	a