拟南芥基因转移新方法一真空渗入法的研究

STUDIES ON A NEW GENE TRANSFORMATION METHOD:VACUUM INFILTRATION TRANSFORMATION

以拟南芥（Ａｒａｂｉｄｏｐｓｉｓｔｈａｌｉａｎａ）生态型Ｌａｎｄｓｂｅｎｇｅｒｅｃｔａ为试材，在含有所构建的ＣａＭＶＢａｒｉ－１株系基因ＶＩ的质粒（ｐＪＯ５３０Ｂａｒｉ－１ＧＶＩ）的根癌农杆菌（Ａｇｒｏｂａｃｔｅｒｉｕｍｔｕｍｅｆａｃｉｅｎｓ）菌种ＧＶ３１０１的介导下，研究了基因转移的新方法一真空渗入法。这种方法简便、快速、可靠且无需经过组织培养阶段即可获得大量转化植株。适宜的转化条件是将生长健壮，除去主苔后４－８ｄ的成株连同营养钵倒置浸入被渗入培养基稀释的农杆菌孢子悬浮液，其光密度为０．８，在吸力为１．７ｍ２／ｈ的真空泵下断续处理２ｍｉｎ／３０ｓ，置于２４ｈ连续光照下的气候室培养，待种子收获后在含有潮霉素的选择培养基选择转化植株．ＰＣＲ分析及ＥＬＩＳＡ检测，该方法的转化效果高达０．７１％。

A new gene transformation method,vacuum infiltration transformation was studiedby the GV3101 strain of Agrobacterium tumefaciens carrying the construction plasmidpJo530:: Bari- 1 GVI in Arabidopsis thaliana Hegn.,ecotype Landsberg erecta. Manytransgenic plants were obtained by this simple,convenient, speedy,reliable and nontissue cultural method. The suitable conditions of vacuum infiltration method was as follows. The robust plants were cultured in the greenhouse. Emerging bolts were clippedoff to encourage growth of multiple secondary bolts. The infiltration would be done fourto eight days after clipping. The agrobacterium carrying pJo530:: Bari-1 GVI wasgrown,centrifuged and then resuspended in infiltration medium (1/2 × MS, 1 × B5 vitamins, 5. 0% sucrose,0. 04 μmol/L Benzylamino purine) for a final OD600 approximate 0.8. The agrobacterium(in infiltration medium) was added to a dish and the plants(pot,soil and all)were inverted into it,and then to place them into bell jar of vacuum pump ofwhich sauction capacity was 1. 7 m2/h. The infiltrated plants would be removed to greenhouse for continuous culture at continuous light until the time of vacuum infiltrationlasted for 2 min/30 s (Drawing a vacuum for 2 minutes, then releasing vacuum veryrapidly, and closing the pump valve to draw a vacuum for another 30 seconds). Whensiliques on plants were very dry,the seed were harvested and were selected on selectionplates (1/2 × MS, 0. 8% agar, 1× B5 vitamins and 30 μg/ml hygromycin). The frequence of transformants was as high as 0. 71% by the analysis of PCR and ELISA.