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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2018, Vol. 38 Issue (1): 25-31    DOI: 10.13523/j.cb.20180103
研究报告     
百合鳞片的诱导分化及遗传转化效率分析
安婷1,季静1(),王昱蓉2,3,马志刚4,王罡1(),李倩1,杨丹1,张松皓1
1 天津大学环境科学与工程学院 天津 300072
2 天津市天大天福生物技术有限公司 天津 300072
3 加州大学圣地亚哥分校 圣地亚哥 92093
4 天津大学化学工程与技术学院 天津 300072
Analysis of the Transformation Efficiency and Induced Differentiation of Lilium brownii Scales
Ting AN1,Jing JI1(),Yu-rong WANG2,3,Zhi-gang MA4,Gang WANG1(),Qian LI1,Dan YANG1,Song-hao ZHANG1
1 School of Environmental Science and Engineering,Tianjin University, Tianjin 300072, China
2 Tianda Tianfu Bio.Co.LTD, Tianjin 300072, China
3 UCSD, Biological Sciences, Molecular Biology, California 92093, USA
4 School of Chemical Engineering and Technology, Tianjin University ,Tianjin 300072,China
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摘要：

基因工程是改良百合性状的重要手段,建立高效稳定的遗传转化体系是百合转基因研究的基础。以百合地下茎鳞片为外植体,筛选并优化百合的直接和间接再生体系;把含枸杞GR(Glutathione reductase)基因和筛选基因NPTII的载体,利用农杆菌转化法对鳞片和愈伤组织进行转基因操作,采用正交试验,优化转化条件以建立适合不同受体的遗传转化体系。结果表明,各阶段最优培养条件分别为:鳞片诱导和膨大MS+2mg/L 2,4-D(2,4-二氯苯氧乙酸)+0.1mg/L NAA(萘乙酸)+ 90g/L蔗糖;鳞片直接分化MS+1.0mg/L 6-BA(6-苄氨基嘌呤)+0.2mg/L NAA+ 30g/L蔗糖,间接分化MS+2.5mg/L 2,4-D +0.4mg/L TDZ(噻重氮苯基脲)+60g/L蔗糖;百合鳞片的Kana(卡那霉素)选择压为100mg/L,愈伤组织75mg/L。遗传转化体系条件为:鳞片,农杆菌OD600=0.6,预培养3d,侵染40min,As(乙酰丁香酮)200μmol/L,阳性植株转化率为17.50%;鳞片分化愈伤组织,农杆菌OD600,预培养5d,侵染40min,As 200μmol/L,阳性植株转化率为12.60%。
关键词： 百合;  遗传转化;  GR;  再生体系;  直接分化    
Abstract:

Genetic engineering is an important means to improve the traits of lily. To establish an efficiency and stable transformation system is the basis of transgenic research. The different regeneration systems of lily were screened. The vector containing the target gene GR and selection gene NPTII was screened by Agrobacterium tumefaciens. Establish efficiency genetic transformation system for callus and scales through orthogonal test. The results showed that the optimum culture conditions as follow. The medium of lily scale inducing was MS+2mg/L 2,4-D + 0.1mg/L NAA + 90g/L + 0.2mg/L NAA + 30g/L sucrose. Indirect differentiation medium was MS+2.5mg/L 2,4-D+0.4mg/L 0.4mg/L TDZ+60g/L sucrose. The Kana selection pressure for scales was 100mg/L and for callus was 75mg/L. The direct genetic transformation of the scales was Agrobacterium OD600 = 0.6, pre-culture for 3 days, infiltrating for 40min, As 200μmol/L. The indirect callus transformation was: Agrobacterium OD600, pre-incubated for 5 days, infiltrating for 40min, As 200μmol/L. The direct and indirect positive plant transformation rates were 17.50% and 12.60%.
Key words: Lily    Genetic transformation    GR    Regeneration system    Direct differentiation
收稿日期: 2017-07-14 出版日期: 2018-01-31
ZTFLH:  Q813  
基金资助: 国家自然科学基金(31271793);国家科技重大专项资助项目(2014Z1-0002)
作者简介: 通讯作者 季静。E-mail: jijingtjdx@163.com
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安婷
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杨丹
张松皓

引用本文:

安婷,季静,王昱蓉,马志刚,王罡,李倩,杨丹,张松皓. 百合鳞片的诱导分化及遗传转化效率分析[J]. 中国生物工程杂志, 2018, 38(1): 25-31.

Ting AN,Jing JI,Yu-rong WANG,Zhi-gang MA,Gang WANG,Qian LI,Dan YANG,Song-hao ZHANG. Analysis of the Transformation Efficiency and Induced Differentiation of Lilium brownii Scales. China Biotechnology, 2018, 38(1): 25-31.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20180103    或    https://manu60.magtech.com.cn/biotech/CN/Y2018/V38/I1/25

NPTII基因引物序列GR基因引物序列
引物名称引物序列引物名称引物序列
NPTII-F5'-GCTATGACTGGGCACAACAG-3'LcGR F5'-TCGTAATAGGTGCTGGAAGTGGT-3'
NPTII-R5'-AAGGAGCACGAAATGCCATA-3'LcGR R5'-ATGTGCTTCGCCGAATAGGTTA-3'
表1  各基因PCR检测引物序列
图1  pCAMBIA2300-LcGR载体图
编号2,4-D
(mg /L)		NAA
(mg /L)		蔗糖
(g /L)		增殖分
化率(%)
1103030 e
210.16040 de
310.29045 d
4206075 c
520.190100 a
620.23070 c
7306085 bc
830.13075 c
930.29090 ab
表2  不同浓度的2,4-D、NAA和蔗糖对鳞片的增殖影响
图2  百合再生过程
编号6-BA
(mg /L)		NAA
(mg /L)		分化率(%)生长状态
10.50.270 bc浅绿,较弱
20.50.480 b浅绿,较弱
30.50.680 b浅绿,健康
410.295 a深绿,强壮
510.480 b深绿,健康
610.665 c深绿,较慢
71.50.265 c浅绿,健康
81.50.450 d细弱,缓慢
91.50.650 d细弱,缓慢
表3  不同浓度的6-BA和NAA对鳞片不定芽形成的影响
编号2,4-D
(mg /L)		TDZ
(mg /L)		蔗糖
(g /L)		分化率(%)
120.13030 f
220.26070 e
320.39089 ab
42.50.46092 a
52.50.69070 e
62.50.23070 e
730.46085 bc
830.23075 de
930.69080 cd
表4  不同浓度2,4-D、TDZ和蔗糖对鳞片愈伤诱导的影响
Kan浓度(mg /L)鳞片褐化率(%)愈伤组织褐化率(%)
000
2522.426.1
5049.264.3
7568.892.3
10095.7100
125100100
表5  受体组织的抗生素敏感试验
序 号农杆菌
OD值		预培养
时间(d)		侵染时
间(min)		As浓度
(μmol/L)		分化率
(%)		最终转化
率(%)
10.41201009.64.69 f
20.433015010.686.52de
30.454020019.29.73 b
40.613015010.687.59cd
50.634020030.2012.9 a
60.652010020.309.21bc
70.813015012.907.67cd
80.832010010.405.84ef
90.854020013.407.91cd
表6  鳞片转化条件优化
序号农杆菌
OD值		预培养
时间(d)		侵染时
间(min)		As浓度
(μmol/L)		分化率
(%)		最终转化
率(%)
10.412010012.802.10 d
20.433015031.007.05 bc
30.454020032.0011.6 a
40.613015012.404.58 cd
50.634020025.509.21 bc
60.652010020.208.20 bc
70.81301509.204.06 cd
80.832010011.27.83 cd
90.854020016.308.13 bc
表7  愈伤转化条件优化
图3  遗传转化
图4  转基因百合的NPT II(a)和LcGR(b)基因的PCR产物电泳图
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