Agrobacterium tumefaciens-mediated transformation of hybrid sweetgum embryogenic callus
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摘要:
目的 杂交枫香是我国重要的用材和观赏树种资源，但其遗传转化体系尚未建立。建立杂交枫香遗传转化体系为杂交枫香性状改良和基因功能研究提供了方法。 方法 本研究基于杂交枫香高效的体细胞胚胎发生技术，用根癌农杆菌介导的遗传转化法对其胚性愈伤组织进行遗传转化，对潮霉素选择压、菌液浓度、侵染时间、共培养时间、以及共培养温度等影响因素采用正交试验等设计进行了研究。 结果 潮霉素对胚性愈伤组织的最小致死质量浓度为10 mg/L；获得最多Gus阳性斑点数的组合的菌液OD600为0.8，共培养时间为3 d，侵染时间为10 min，共培养温度为25 ℃；获得最多转基因阳性愈伤组织的组合的菌液OD600为0.2，共培养时间为2 d，侵染时间为10 min，共培养温度为23 ℃；且通过极差分析，最优处理组合的共培养时间为2 d，菌液OD600为0.2，侵染时间为20 min，共培养温度为23 ℃。 结论 经分子鉴定，共获得210个转基因阳性愈伤组织，初步建立了农杆菌介导的杂交枫香遗传转化体系，为阔叶树种愈伤组织的遗传转化提供了更多的可行性依据。 Abstract:Objective Hybrid sweetgum is an important timber and ornamental tree resources in China, but its genetic transformation system has not been established yet. Establishing genetic transformation system of hybrid sweetgum provides a useful approach for trait improvement and allows us to conduct a functional identification of gene in hybrid sweetgum. Method Based on the efficient somatic embryogenesis of hybrid sweetgum, the embryogenic calluses were transformed by Agrobacterium tumefaciens-mediated genetic transformation. Factors influencing transformation were studied by orthogonal experiment, including hygromycin selective pressure, concentration of Agrobacterium, infection time, co-culture time and co-culture temperature. Result Results showed that the minimum lethal concentration of hygromycin to embryogenic callus was 10 mg/L. The number of Gus positive spots was highest when bacterium solution (OD600) was 0.8, co-culture time was 3 d, infection time was 10 min, co-culture temperature was 25 ℃. The most transgenic positive resistant calluses were obtained when bacterium solution (OD600) was 0.2, co-culture time was 2 d, infection time was 10 min, co-culture temperature was 23 ℃. And the optimal treatment combination was 0.2 bacterium solution (OD600), 2 d co-culture time, 20 min infection time, 23 ℃ co-culture temperature. Conclusion A total of 210 positive transgenic callus were obtained by molecular identification. The genetic transformation system was established, which provides more feasible foundation for the transformation of broadleaf tree callus. -
Key words:
- hybrid sweetgum /
- embryogenic callus /
- Agrobacterium tumefaciens /
- genetic transformation
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图 2 愈伤鲜质量增长率随培养天数和潮霉素质量浓度的变化
Figure 2. Variation of callus fresh mass proliferation rate with incubation days and hygromycin mass concentration
图 3 Gus在愈伤组织中7 d后的表达情况
A、B. 菌液OD600 = 0.2时，Gus表达情况；C. 菌液OD600 = 0.5时，Gus表达情况；D. 菌液OD600 = 0.8时，Gus表达情况。A, B, concentration of bacterium OD600 = 0.2, the expression of Gus; C, concentration of bacterium OD600 = 0.5, the expression of Gus; D, concentration of bacterium OD600 = 0.8, the expression of Gus.
Figure 3. Expression of Gus in callus after 7 d
图 4 抗性愈伤组织
A. 抗性愈伤组织在筛选培养基上生长；B. 抗性愈伤的进一步筛选。A, antibiotic-resistant callus formed on selective medium; B, develepment of antibiotic-resistant cell lines for further selection.
Figure 4. Antibiotic-resistant callus
图 5 Hyg抗性细胞系PCR检测
A. Gus 453 bp；B. hpt 557 bp；M. Trans2K Plus DNA Marker；W. 野生型；1-10、2-12、418、5-7、7-11、9-20. 转基因愈伤组织；+. 质粒阳性对照。下同。W, wild type；1−10, 2−12, 4−18, 5−7, 7−11, 9−20, transformed cell lines; +, plasmid for positive control. The same below.
Figure 5. PCR analysis of hygromycin-resistant embryogenic cell line
图 6 Hyg抗性细胞系VirD2基因PCR检测
Figure 6. PCR analysis of VirD2 gene about hygromycin-resistant embryogenic cell line
表 1 实验所用培养基
Table 1. Culture medium used in this research
培养基 Culture medium 成分 Component LB固体培养基
LB solid medium5 g/L酵母抽提物 + 10 g/L胰蛋白胨 + 10 g/L NaCl + 50 mg/L Kan + 15 g/L 琼脂粉
5 g/L yeast extract + 10 g/L tryptone + 10 g/L NaCl + 50 mg/L Kan + 15 g/L agar液体增殖培养基
Liquid propagation medium基本培养基[13] + 40 g/L蔗糖 + 1 g/L酶水解酪蛋白，pH调至5.6 ~ 5.7
Basal medium[13] + 40 g/L sucrose + 1 g/L casein hydrolysate, adjust pH to 5.6 −5.7固体增殖培养基
Solid propagation medium基本培养基[13] + 40 g/L蔗糖 + 1 g/L酶水解酪蛋白 + 3 g/L植物凝胶，pH调至5.6 ~ 5.7
Basal medium[13] + 40 g/L sucrose + 1 g/L casein hydrolysate + 3 g/L phytagel, adjust pH to 5.6 − 5.7共培养培养基
Co-culture medium基本培养基 + 40 g/L蔗糖 + 1 g/L酶水解酪蛋白 + 3 g/L植物凝胶 + 50 μmol/L AS，pH调至5.2
Basal medium[13] + 40 g/L sucrose + 1 g/L casein hydrolysate + 3 g/L phytagel + 50 μmol/L AS, adjust pH to 5.2恢复培养基
Recovery medium基本培养基[13] + 40 g/L蔗糖 + 1 g/L酶水解酪蛋白 + 3 g/L植物凝胶 + 300 mg/L Cef，pH调至5.6 ~ 5.7
Basal medium[13] + 40 g/L sucrose + 1 g/L casein hydrolysate + 3 g/L phytagel + 300 mg/L Cef, adjust pH to
5.6−5.7筛选培养基
Screening medium基本培养基[13] + 40 g/L蔗糖 + 1 g/L酶水解酪蛋白 + 3 g/L植物凝胶 + 300 mg/L Cef + 10 mg/L Hyg，pH调至
5.6 ~ 5.7
Basal medium[13] + 40 g/L sucrose + 1 g/L casein hydrolysate + 3 g/L phytagel + 300 mg/L Cef + 10 mg/L Hyg, adjust pH to 5.6−5.7
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表 2 PCR反应体系
Table 2. PCR reaction system
成分 Component 体积 Volume/μL 2 × PCR master mix 10.0 ddH2O 5.2 ~ 7.2 上游引物 Forward primer 0.4 下游引物 Reverse primer 0.4 模板 DNA Template DNA 2.0 ~ 4.0 总体积 Total volume 20.0
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表 3 农杆菌介导转化试验设计及转化结果
Table 3. Experimental design and results of Agrobacterium-mediated transformation
编号
No.共培养时间
Co-culture
time/dOD600
侵染时间
Infection
time/min共培养温度
Co-culture
temperature/℃Gus阳性斑点数/(个· mg−1)
Gus positive spot/
(number· mg−1)抗性愈伤/(个·g−1)
Resistant callus/
(number·g−1)PCR阳性愈伤/(个·g−1)
PCR positive callus/
(number·g−1)1 2 0.2 10 23 0.57Bc 52 50 2 2 0.5 20 25 5.41Bbc 41 39 3 2 0.8 30 28 10.40Bbc 0 0 4 3 0.2 20 28 1.20Bc 40 32 5 3 0.5 30 23 13.07Bb 13 12 6 3 0.8 10 25 26.85Aa 0 0 7 4 0.2 30 25 7.54Bbcc 21 19 8 4 0.5 10 28 9.06Bbc 0 0 9 4 0.8 20 23 14.91Ab 44 28 注：同一列中不同大写字母表示差异极显著(P < 0.01)；同一列中不同小写字母表示差异显著(P < 0.05)。下同。Notes：different capital letters in a column mean very significant difference at P < 0.01 level; different lowercase letters in a column mean significant differences at P < 0.05 level. The same below.
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表 4 不同条件下Gus 阳性斑点数方差分析
Table 4. Variance analysis of Gus positive spots under different conditions
变异来源 Source of variation 自由度 df 均方 MS F值 F value 显著性 Sig. 共培养天数 Co-culture time 2 155.590 5.111 0.017* OD600 2 462.420 15.189 0.000** 侵染时间 Infection time 2 57.321 1.883 0.181 共培养温度 Co-culture temperature 2 92.635 3.043 0.073 误差 Error 18 30.444 总计 Total 26 注：**表示影响极显著(P < 0.01)，*表示影响显著(P < 0.05)。Notes: ** represents significant influence (P < 0.01); * represents very significant influence (P < 0.05).
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表 5 不同条件下Gus阳性斑点数极差分析
Table 5. Range analysis of Gus positive spots under different conditions
指标
Index各因素不同条件下Gus阳性斑点数/(个·mg−1)
Gus positive spots detected at different gradients
of each factor/(number·mg−1)共培养时间
Co-culture timeOD600 侵染时间
Infection time共培养温度
Co-culture temperatureK1 5.46 3.10 12.16 9.52 K2 13.71 9.18 7.17 13.27 K3 10.50 17.39 10.34 6.89 R 8.25 14.29 4.99 6.38 注： 1表示各因素水平分别为共培养时间为2 d，菌液OD600为0.2，侵染时间为10 min，共培养温度为23 ℃；2表示各因素水平分别为共培养时间为3 d，菌液OD600为0.5，侵染时间为20 min，共培养温度为25 ℃；3表示各因素水平分别为共培养时间为4 d，菌液OD600为0.8，侵染时间为10 min，共培养温度为28 ℃。下同。 Notes: 1 means that the level of each factor is 2 d co-culture time, 0.2 bacterium solution (OD600), 10 min infection time, 23 ℃ co-culture temperature respectively; 2 means that the level of each factor is 3 d co-culture time, 0.5 bacterium solution (OD600), 20 min infection time, 25 ℃ co-culture temperature respectively; 3 means that the level of each factor is 4 d co-culture time, 0.8 bacterium solution (OD600), 30 min infection time, 28 ℃ co-culture temperature respectively. The same below.
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表 6 不同条件下阳性愈伤组织数极差分析
Table 6. Range analysis of the number of positive calluses under different conditions
指标
Index各因素不同梯度时阳性愈伤组织数(个·g−1)
Number of positive calluses detected at different
gradients of each factor/(number·g−1)共培养时间
Co-culture timeOD600 侵染时间
Infection time共培养温度
Co-culture temperatureK1 89 101 50 90 K2 44 51 99 58 K3 47 28 31 32 R 45 73 68 58
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