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杂交枫香胚性愈伤组织遗传转化体系研究

江帅菲 崔莹 赵瑞瑞 齐帅征 KongLisheng 赵健 李珊珊 张金凤

江帅菲, 崔莹, 赵瑞瑞, 齐帅征, KongLisheng, 赵健, 李珊珊, 张金凤. 杂交枫香胚性愈伤组织遗传转化体系研究[J]. 北京林业大学学报, 2021, 43(8): 9-17. doi: 10.12171/j.1000-1522.20210032
引用本文: 江帅菲, 崔莹, 赵瑞瑞, 齐帅征, KongLisheng, 赵健, 李珊珊, 张金凤. 杂交枫香胚性愈伤组织遗传转化体系研究[J]. 北京林业大学学报, 2021, 43(8): 9-17. doi: 10.12171/j.1000-1522.20210032
Jiang Shuaifei, Cui Ying, Zhao Ruirui, Qi Shuaizheng, Kong Lisheng, Zhao Jian, Li Shanshan, Zhang Jinfeng. Agrobacterium tumefaciens-mediated transformation of hybrid sweetgum embryogenic callus[J]. Journal of Beijing Forestry University, 2021, 43(8): 9-17. doi: 10.12171/j.1000-1522.20210032
Citation: Jiang Shuaifei, Cui Ying, Zhao Ruirui, Qi Shuaizheng, Kong Lisheng, Zhao Jian, Li Shanshan, Zhang Jinfeng. Agrobacterium tumefaciens-mediated transformation of hybrid sweetgum embryogenic callus[J]. Journal of Beijing Forestry University, 2021, 43(8): 9-17. doi: 10.12171/j.1000-1522.20210032

杂交枫香胚性愈伤组织遗传转化体系研究

doi: 10.12171/j.1000-1522.20210032
基金项目: 国家林业和草原局推广项目(2020133102),中央高校基础研究基金项目(2015ZCQ-SW-02),河南省许昌市重大科技专项(20170112006)
详细信息
    作者简介:

    江帅菲。主要研究方向:杂交枫香遗传转化及基因编辑。Email:435414605@qq.com 地址:100083 北京市海淀区清华东路35号北京林业大学生物科学与技术学院

    责任作者:

    张金凤,教授,博士生导师。主要研究方向:树木体胚胎发生与倍性育种。Email:zjf@bjfu.edu.cn 地址:同上

  • 中图分类号: S722.3

Agrobacterium tumefaciens-mediated transformation of hybrid sweetgum embryogenic callus

  • 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。

     

  • 图  1  杂交枫香胚性愈伤组织

    Figure  1.  Embryogenic callus of Liquidambar styraciflua × L. formosana

    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 medium
    5 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
    下载: 导出CSV

    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
    下载: 导出CSV

    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/d
    OD600

    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)
    120.210230.57Bc5250
    220.520255.41Bbc4139
    320.8302810.40Bbc00
    430.220281.20Bc4032
    530.5302313.07Bb1312
    630.8102526.85Aa00
    740.230257.54Bbcc2119
    840.510289.06Bbc00
    940.8202314.91Ab4428
    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.
    下载: 导出CSV

    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 MSFF 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).
    下载: 导出CSV

    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 time
    OD600侵染时间
    Infection time
    共培养温度
    Co-culture temperature
    K1 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.
    下载: 导出CSV

    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 time
    OD600侵染时间
    Infection time
    共培养温度
    Co-culture temperature
    K1891015090
    K244519958
    K347283132
    R45736858
    下载: 导出CSV
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出版历程
  • 收稿日期:  2021-01-29
  • 修回日期:  2021-02-22
  • 网络出版日期:  2021-07-15
  • 刊出日期:  2021-08-31

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