农杆菌介导的杜仲叶片愈伤组织遗传转化体系

doi:10.11707/j.1001-7488.20200209

摘要/Abstract

摘要：

目的: 优化杜仲叶片愈伤组织的再生体系，研究愈伤组织对抗生素和抑菌剂的敏感性，探索影响农杆菌介导杜仲愈伤组织遗传转化的最适转化因子水平，构建农杆菌介导的杜仲愈伤组织瞬时转化体系，使杜仲成年植株外植体为受体的遗传转化成为可能，为杜仲基因功能的研究与定向改良奠定基础。方法: 以杜仲成年植株叶片为材料诱导愈伤组织，通过添加不同浓度植物生长调节剂与大量元素的MS培养基进行不定芽的诱导与增殖，确定最适培养基。在此基础上，在培养基中添加不同浓度抗生素及抑菌剂，研究愈伤组织对其敏感性。以获得的叶片愈伤组织受体系统为基础，通过L₁₆（4⁵）的正交试验，探索不同转化因子对农杆菌介导叶片愈伤组织遗传转化的瞬时转化效率的影响，建立最适瞬时转化体系。使用获得的瞬时转化体系对愈伤组织进行遗传转化操作，筛选抗性芽，对抗性芽进行GUS组织化学染色与PCR检验。结果: 再生体系优化的结果表明，3/4大量元素浓度的MS培养基能够促进杜仲愈伤组织不定芽的诱导及生长；愈伤组织诱导不定芽的最适培养基为3/4MS+0.27 μmol·L^-1 NAA+4.4 μmol·L^-1 6-BA，诱导率为83%±10.0%；不定芽复壮的最适培养基为3/4MS+0.054 μmol·L^-1 NAA+4.4 μmol·L^-1 6-BA，平均伸长长度为（2.47±1.33）cm。抗生素与抑菌剂敏感性试验表明，遗传转化的选择培养基中，抑菌剂头孢霉素的最适浓度为200 mg·L^-1，筛选用的抗生素卡那霉素最适浓度为70 mg·L^-1。转化因子的正交试验表明，最适的农杆菌介导杜仲叶片愈伤组织遗传转化的转化因子组合为：预培养5天、侵染10 min和共培养3天。使用最适瞬时转化体系对约200个愈伤组织进行遗传转化操作，共筛选获得3个抗卡那霉素的抗性芽；GUS组织化学染色显示GUS基因在抗性芽中得到了表达，PCR检测证明这些抗性芽中存在NPTⅡ基因。结论: 杜仲叶片愈伤组织诱导不定芽的最适培养基为3/4MS+0.27 μmol·L^-1 NAA+4.4 μmol·L^-1 6-BA，不定芽复壮的最适培养基为3/4MS+0.054 μmol·L^-1 NAA+4.4 μmol·L^-1 6-BA。农杆菌介导的杜仲叶片愈伤组织瞬时转化体系为：预培养5天、侵染10 min和共培养3天，筛选培养基为3/4MS+0.054 μmol·L^-1 NAA+4.4 μmol·L^-1 6-BA+200 mg·L^-1 Cef+70 mg·L^-1 Km。利用此体系共获得3个抗性芽，PCR分析和GUS组织化学染色都表明T-DNA已整合到抗性芽基因组中。

关键词: 杜仲, 农杆菌介导遗传转化, 再生体系, GUS瞬时表达

Abstract:

Objective: To optimize the callus regeneration system for Eucommia ulmoides leaf explants, to assess the sensitivity of callus to Km and Cef, and to determine the optimum factor levels for Agrobacterium tumefaciens-mediated transformation, in order to allow for genetic transformation of adult plant of E. ulmoides. Method: Callus was induced from leaves of E. ulmoides adult plants. It was inoculated in different concentrations of MS medium, NAA and 6-BA to optimize culture conditions for adventitious bud induction and regeneration. In addition, different concentrations of Km and Cef were added to the culture medium to study callus sensitivity. Based on the obtained callus receptor system for leaves, an L₁₆ (4⁵) orthogonal array experiment was conducted to explore the effect of different conversion factors on Agrobacterium-mediated transformation efficiency. Transformation with an optimum combination of factors was used to obtain Km-resistant buds, which were assessed as transformants by GUS histochemical staining and PCR. Result: MS medium at 3/4 strength promoted the induction and growth of adventitious buds in E. ulmoides. The optimum medium for induction of adventitious buds in callus was 3/4MS + 0.27 μmol·L^-1 NAA + 4.4 μmol·L^-1 6-BA, with induction rate of 83%±10.0%; The optimal medium for adventitious bud rejuvenation was 3/4MS + 0.054 μmol·L^-1 NAA + 4.4 μmol·L^-1 6-BA, with an average growth length of (2.47 ±1.33) cm. Sensitivity testing of antibiotic and bacteriostatic agents showed that in the selected medium for genetic transformation, the optimal concentrations of Cef and Km for screening were 200 mg·L^-1 and 70 mg·L^-1, respectively. The orthogonal test showed that the optimum combination of factors for Agrobacterium-mediated transformation was 5 days of pre-culture, 10 min of infection time and 3 days of co-cultivation. Genetic transformation of about 200 calli was carried out using the optimum instantaneous conversion system, and three Km-resistant buds were obtained. GUS histochemical staining showed that the GUS gene was expressed in these Km-resistant buds, and PCR analysis demonstrated the presence of the NPTⅡ gene. Conclusion: For E. ulmoides callus, this study defined an adventitious bud induction medium, 3/4MS+0.27 μmol·L^-1 NAA + 4.4 μmol·L^-1 6-BA, and an adventitious bud rejuvenation medium:3/4MS + 0.054 μmol·L^-1 NAA + 4.4 μmol·L^-1 6-BA. Agrobacterium-mediated transformation of callus involved 5 days of pre-culture, 10 min of infection time and 3 days of co-cultivation, and the selection medium, 3/4MS + 0.054 μmol·L^-1 NAA + 4.4 μmol·L^-1 6-BA + 200 mg·L^-1 Cef + 70 mg·L^-1 Km. Three resistant buds were obtained using this system. PCR analysis and GUS histochemical staining indicated that T-DNA had been integrated into the genome of these Km-resistant buds. This report demonstrated that genetic transformation could be studied in a mature E. ulmoides tree, laying a foundation for studying gene functions and for objective-specific improvement of E. ulmoides.

Key words: Eucommia ulmoides, Agrobacterium-mediated transformation, regeneration process, GUS transient expression

中图分类号:

S718.46

刘闵豪,徐郡儡,叶靖,李周岐,范睿深,李龙. 农杆菌介导的杜仲叶片愈伤组织遗传转化体系[J]. 林业科学, 2020, 56(2): 79-88.

Minhao Liu,Junlei Xu,Jing Ye,Zhouqi Li,Ruishen Fan,Long Li. Agrobacterium tumefaciens-Mediated Transformation of Leaf Callus in Eucommia ulmoides[J]. Scientia Silvae Sinicae, 2020, 56(2): 79-88.

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培养基 Medium	6-BA浓度 Concentration of 6-BA/ (μmol·L^-1)	NAA浓度 Concentration of NAA/(μmol·L^-1)	不定芽诱导率 Percentage of shoot induction (%) (mean± SD)	不定芽伸长长度 Shoot length/cm (mean ± SD)
MS	0	0.27	0±0 f	—
MS	0	0.54	0±0 f	—
MS	4.4	0.054	—	0.51±0.48 b
MS	4.4	0.27	53±7.0 abcd	0.86±0.91 ab
MS	4.4	0.54	20±8.7 ef	-
MS	6.7	0.27	48±15.6 bcde	-
MS	8.8	0	24±9.1 def	-
MS	8.8	0.054	—	0.34±0.41 b
MS	8.8	0.27	19±4.5 ef	0.48±0.27 b
MS	8.8	0.54	55±14.4 abc	-
3/4MS	0	0.27	0±0 f	-
3/4MS	0	0.54	0±0 f	-
3/4MS	4.4	0.054	—	2.47±1.33 a
3/4MS	4.4	0.27	83±10.0 a	1.39±1.01 ab
3/4MS	4.4	0.54	43±10.7 bcde	-
3/4MS	6.7	0.27	60±16.8 ab	-
3/4MS	8.8	0	47±21.9 bcde	-
3/4MS	8.8	0.054	-	1.21±0.65 ab
3/4MS	8.8	0.27	26±6.0 cdef	1.03±0.82 ab
3/4MS	8.8	0.54	66±11.0 ab	-

编号 No.	Km浓度 Concentration of Km/(mg·L^-1)	Cef浓度 Concentration of Cef/(mg·L^-1)	不定芽诱导数 Number of shoot induced	愈伤组织状况 Growing states of callus
1	0	0	21	黄绿色,致密Yellowish green, compact
2	20	0	15	黄绿色,致密Yellowish green, compact
3	50	0	0	黄褐色,致密Yellowish brown, compact
4	70	0	0	褐色,死亡Brown, dead
5	100	0	0	褐色,死亡Brown, dead
6	0	0	18	黄褐色,致密Yellowish brown, compact
7	0	100	24	黄褐色,致密Yellowish brown, compact
8	0	200	20	黄褐色,致密Yellowish brown, compact
9	0	300	5	黄褐色,致密Yellowish brown, compact
10	0	400	0	褐色,死亡Brown, dead

方差来源 Sources of variance	平方和 Sum of squares	自由度 Degree of freedom	均方 Mean square	F	P
A:预培养时间Pre-culture duration	0.082	3	0.027	7.746	0.063
B:AS浓度AS concentration	0.003	3	0.001	0.262	0.850
C:侵染时间Infection duration	0.102	3	0.034	9.658	0.047^*
D:共培养时间Co-culture duration	0.281	3	0.094	26.575	0.012^*
误差Error	0.011	3	0.004
总计Total	1.618	16

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