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林业科学 ›› 2002, Vol. 38 ›› Issue (2): 92-97.doi: 10.11707/j.1001-7488.20020216

• 论文及研究报告 • 上一篇    下一篇

柞蚕抗菌肽D基因转化桉树培育抗青枯病株系的研究

邵志芳 陈伟元 罗焕亮 叶新丰 张景宁   

  1. 深圳市莲花山公园管理处,深圳518036;深圳市梧桐山苗圃总场,深圳518114;深圳出入境检验检疫局,深圳518045华南理工大学食品与生物工程学院在职博士生;广东省肇庆林业局,肇庆526040;华南农业大学,广州510642
  • 收稿日期:2000-03-05 修回日期:1900-01-01 出版日期:2002-03-25 发布日期:2002-03-25

STUDIES ON THE INTRODUCTION OF THE CECROPIN D GENE INTO EUCALYPTUS UROPHYLLA TO BREED THE RESISTANT VARIETIES TO PSEUDOMONAS SOLANACEARUM

Shao Zhifang,Chen Weiyuan,Luo Huanliang,Ye Xinfeng,Zhang Jingning   

  1. Shenzhen Lianhuashan Park Shenzhen 518036;Shenzhen Wutongshan Nurseries Shenzhen 518114;Shenzhen Inspection and Quarantine Bureau Shenzhen 518045;Zhaoqing Forestry Bureau Zhaoqing 526040;South China Agricultural University Guanzhou 510642
  • Received:2000-03-05 Revised:1900-01-01 Online:2002-03-25 Published:2002-03-25

摘要:

通过二次正交旋转组合设计对尾叶桉叶盘愈伤组织分化培养基优化,获得尾叶桉(Eucalyptus urophylia)叶盘外植体的再生植株。将携带外源目的基因的根癌农杆菌(Agrobacterium tumefaciens)与尾叶桉U6无性系叶盘共培养,经愈伤组织诱导、卡那霉素筛选和植株再生,获得20个小芽,再将小芽转入附加卡那霉素(Kam) 40μg·mL-1 的E3-B培养基中诱根,获得8株存活且可再生的转化子。取转化苗叶片作胭脂碱合成酶活性检测电泳后呈阳性;分别以γ-32p-dCTP及地高辛标记柞蚕抗菌肽D基因作探针,与转化苗DNA作点杂交及Southern印迹杂交检测,结果均呈阳性。以上结果表明,柞蚕抗菌肽D基因已整合到尾叶桉基因组中。将从桉树青枯病重病区分离的强毒青枯菌(Pseudomonas spp .)株(991016)以1×109cfu·mL-1 浓度接种转化试管苗,以未经转基因的尾叶桉U6 无性系试管苗为对照,结果表明转化苗接种死亡率56.7% ,对照死亡率为86.7% ,由此可推出导入柞蚕抗菌肽D基因的尾叶桉转化苗明显提高了对青枯菌的抗性。

关键词: 尾叶桉, 抗菌肽D, 青枯病, 柞蚕

Abstract:

The leaf disc of Eucalyptus urophylla was regenerated by use of the quadratic orthogonal rotation design to optimize the differentiation medium for the callus.The leaf discs of the E.urophylla were co-cultivated with Agrobacterium tumefaciens carrying the target gene.20 sprouts which resisted to the Kam were obtained after callus inducing and Kam sereening.8 out of them regenerated from the rooting medium E3-B supplied with the same concentration of Kam can be considered as transgenic seedlings.The nopaline synthetic enzyme activity of the preliminary transformation was detected by electrophoresis.Using the anti-bacterial peptide gene labelled with γ-32p-dCTP or DIG as probes,the introduced gene was detected in the preliminary transformations through dot blotting and Southern blotting.Innoculation of the preliminary transgenic seedlings with high virulent Pseudomonas solanacearum (991016) caused a death rate of 56.7%,30% lower than that of the control.It was demonstrated from the above results that the anti-bacterial peptide gene was integrated into the genome of Eucalyptus plant and the transgenic plant with higher resistance were obtained.

Key words: Eucalyptus urophylla, Cecropin D, Bacterial-wilt disease, Antheraea pernyi