摘要
我们利用自行安装的ＰＩＧ基因枪瞬时转化水稻悬浮细胞，对其操作参数及转化条件的优化进行了研究。ＰＩＧ基因枪结构简单，操作方便，价格及维持费很低。研究结果表明，在设计参数为轰击持续时间５０ｍｓ及样品室真空度为２９～３０ｉｎ．Ｈｇ时，其它操作参数的优化值为氦气压力６０ＰＳｌ，轰击高度为１７ｃｍ，缓冲网板高度为ｇｃｍ。水稻悬浮细胞在轰击前后分别进行高渗处理（含有０．５Ｍ甘露醇的ＬＳ０．５培养基）４ｈ和１０～１６ｈ可提高转化率４倍以上，在ＤＮＡ吸附过程中，用乙醇进行后处理比用水处理可提高转化率３．８倍。另外，用金粉为ＤＮＡ载体比钨粉效果好，转化量可提高一倍以上。这些结果证实ＰＩＧ基因枪的可靠性及稳定性。这对促进ＰＩＧ基因枪在植物基因工程中的应用有着重要意义。
The purpose of this study was to test the parameters and conditions for efficient transient transformation of rice suspension cells using a self-constructed ParticleInflow Gun(PIG).The PIG device was constructed following Dr. John J. Finer's design.The rice variety used was Zhongguo 91 (a japonica variety),and the plasmid used waspACTI-D(containing gusA gene). The results showed that the parameters resulting inthe highest efficiency of transient transformation were as follows: the cells were placed17 cm below the syringe filter unit where the DNA-coated particles were placed,heliumstream of 60PSI was released for 50ms for each bombardment with a vacuum of 28 Hg inthe sample chamber.Also, it was necessary to place a baffle plate gcm above the suspension cells.Different DNA precipitation procedures affected the efficiency of transienttransformation.Using ethanol in DNA precipitation and re-suspension of coated particles gave more than 4-fold increase in transient transformation than using distilled water. Placement of cells on an osmoticum-containing medium(LS0. 5 Containing 0. 5 Mmannitol)4 h prior to and 10-16 h after bombardment also result in 4-fold increase intransient β-glucuronidase expression. Stable transformation of rice was also obtainedusing the PIG device.These results indicate that the PIG device is reliable,and could besuccessfully used in rice transformation.
出处
《山东农业大学学报（自然科学版）》
CSCD
1996年第2期123-128,共6页
Journal of Shandong Agricultural University：Natural Science Edition
基金
国家自然科学基金
关键词
PIG
基因枪
高渗处理
DNA
吸附
水稻
基因转化
Particle Inflow Gunl transient transformation of rice
osmotic treatment,DNA precipitation
