全 文 ：文章编号: 1007-0435( 2005) 02-0172-02 ·博士论文摘要·
冰草组织培养再生体系建立
及耐旱转基因研究
博士生:霍秀文1, 2　导师:云锦凤2 ,米福贵2 ,魏建华3
( 1.内蒙古农业大学农学院, 内蒙古呼和浩特　010019; 2.内蒙古农业大学生态环境学院 ,内蒙古呼和浩特　010019;
3. 北京市农林科学院农业生物技术中心, 北京　100089)
　　本研究以冰草属植物中的 4个品种——蒙
古冰草新品系( A . mongolicum Keng)、航道冰
草( A . cr istatum cv. Fairw ay )、诺丹冰草( A .
deser torum cv. Nordan)和一个种间杂种冰草
—— 蒙 农 杂 种 冰 草 ( A . cristatum × A .
deser torum cv. Hycrest-Mengnong )为材料, 分
别以幼穗为外植体建立了冰草组织培养再生体
系。在此基础上,以调控脯氨酸生物合成最后一
步的关键酶的突变体基因 P5CS 为目标基因,
bar 基因为筛选标记基因, 用基因枪法轰击幼
穗诱导的愈伤组织, 获得转基因植株。利用
PCR方法从拟南芥( A rabidop sis thaliana)基因
组 DNA 中分离得到了 CBF4 及其启动子, 并
对CBF4启动子进行了序列分析和初步的功能
分析。结果表明:
1.冰草幼穗的取样时期为孕穗期, 长度介
于 1. 0～3. 0 cm 的幼穗为最适宜的外植体; 诱
导愈伤组织的最适培养基为改良 MS+ 2, 4-D
2. 0 mg / L , 诱导率 83. 5%; 分化培养基为 MS
+ KT 0. 2 mg/ L , 分化率 74. 5% ;生根培养基
为 1/ 2 M S,生根率 100%。
2. 以幼穗诱导的愈伤组织为受体,采用基
因枪轰击法转化冰草获得转基因植株; PCR和
Southern检测表明外源基因 P5CS 已整合到冰
草属植物基因组 DNA 中; RT -PCR 检测表明
目的基因已在冰草转基因植株的转录水平表
达; P5CS 基因的遗传转化率为 0. 09%～
0. 11%。
3. 采用PCR方法从拟南芥中分离了CBF4
基因及其启动子序列,采用生物信息学方法对
启动子进行序列分析,表明 CBF4 启动子可能
为逆境诱导型启动子。将其与 GUS 基因融合
转化烟草, GU S活性检测初步表明该启动子受
干旱逆境诱导。
关键词:冰草;植株再生; P5CS基因; CBF4; CBF4启动子
中图分类号: Q943. 2　　　文献标识码: A
论文完成时间: 2004年 5月
收稿日期: 2004--;修回日期: 2004--
基金项目:国家转基因植物与产业化研究专项( J00-B-001-13)和( J2002-B-008)资助项目
作者简介:霍秀文( 1968-) ,女,内蒙古呼和浩特人,博士, 副教授, 主要从事园艺植物育种和生物技术与研究, E-mail: huoxiuw en
@ sohu. com .
导师简介:云锦凤( 1941-) ,女,蒙古族,内蒙古土默特旗人,教授,主要从事牧草遗传育和种质资源的教学和科研工作, 发表论文 70
余篇,出版专著 4部,编写教材 2部, E-m ail : csgrass@ publ ic. hh . nm. cn
第 13卷　第 2期
　Vo l. 13　　No. 2
草　地　学　报
ACT A AGRESTIA SIN ICA
　　2005年　 6 月
　June 　　2005
Study on Culture and Regeneration of Wheatgrass and Its Genetic
Transformation for Drought Tolerance
Candidate: HUO Xiu-w en
1, 2　Adviso r: YU N Jin-feng 2, MI Fu-gui2, WEI Jian-hua3
(1. Col lege of Agronomy, Inner M on golia Agricultural University, Huhhot , Inner Mongol ia
Autonomous Region 010019, China;
2. College of Ecology and Environment , Inner Mon gol ia Agricultural University, Huhhot ,
Inner M on golia, Autonomous Region 010019, China;
3. Beijing Agro-Biotechnology Research Cen ter, Beijin g Academy of Agriculture and
Forest Sciences , Bei jing 100089, Chin a)
This paper tells how the w heatgrass t issue culture and regenerat ion system w ith the immature
inf lorescence as explant w as established using four species, namely: A . mongolicum Keng , A . cristatum cv .
Fairw ay, A . desertorum cv. Nordan, and A . cri statum×A . desertor um cv . Hycr est-M engnong as materials.
Based on the established regenerat ion system , the P5CS gene, w hich regulates the last step o f key enzymes
mutant gene o f prol ine synthesis in a plant , w as tr ansformed into w heatgrass w ith phosphino thricin
acetylt ransferase ( bar ) conferring her bicide resistance as select ing gene. The t ransformat ion w as conducted
thr ough microproject ile bombardment of callus derived from immature inflorescence.
The CBF4 gene and its promoter had been isolated from A rabidop sis thaliana by PCR. T he sequence
analy sis and function assay w ere conducted.
The primary results and prog ress are summarized as follows:
1. The inf lorescence of a length of 1. 0 to 3. 0 cm is opt imal to be used as explant to induce the callus. T he
feasible medium for cal lus induct ion from immatur e inf lorescence w as the improved M S+ 2, 4-D ( 2. 0 mg/ L ) .
The induct ion f requency w as above 83. 5%. The dif ferent iat ion medium was M S + KT ( 0. 2 mg / L ) , with a
dif ferent iat ion rat io of 74. 5%. The ro ot ing medium was 1/ 2 MS with 100% roo ting rat io .
2. T he t ransgenic plants w ere obtained by m icroproject ile bombardment of callus induced from immature
inf lorescence, the r esults of PCR and Southern analy sis displayed that the exogenous P5CS gene had integrated
into the genome of t ransgenic w heatgrass, and the assay of RT -PCR showed that the t ransgenic P5CS had
expressed at a transcript lev el . T he tr ansgenic f requencies o f P5CS gene w ere 0. 09%～0. 11% in 2003.
3. T he CBF4 gene and its promoter w ere isolated fr om A rabidop sis thaliana by PCR. The analy sis of CBF4
pr omoter sequence w ith bioinfo rmat ics so ftw are indicated that it could be one o f the inducible pr omoters by
outside env ir onmental st resses. The GU S expression casset te driven by CBF4 promo ter was const ructed and
transfo rmed into tabacco ( Wisconsin 38) . The assay o f the expr ession o f GU S in the t ransgenic tobacco
displayed that CBF4 promoter could be induced by dr ought str ess, in accordance w ith the analysis w ith
bio informat ic sof tw are.
Key words : Wheatgrass ( A gropy ron Gaertn) ; Plant Regener at ion; P5CS Gene; CBF4; CBF 4 Promoter
173第 2期 霍秀文:冰草组织培养再生体系建立及耐旱转基因研究