柞蚕抗菌肽D基因转化桉树培育抗青枯病株系的研究

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

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 (Eucalyptusuro phylia)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 (Agrobacteriumtumefaciens)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 2 0个小芽 ,再将小芽转入附加卡那霉素(Kam) 4 0 μg·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 32 p 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 (Pseudomonasspp .)株 (9910 16 )以 1× 10 9cfu·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 5 6 7% ,对照死亡率为 86 7% 。

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 E 3-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 32 p\|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.