利用共转化方法创制携带Pi9抗稻瘟病基因的无选择标记转基因水稻

水稻稻瘟病是由子囊菌(Magnaporthe oryzae)引起的水稻灾害性病害。培育抗病品种是防治稻瘟病最经济有效的措施之一。水稻Pi9抗稻瘟病基因来源于小粒野生稻并已被克隆和应用于转基因育种。为了提高无选择标记转基因植株的选择效率,将绿色荧光蛋白(GFP)用作可视遗传标记,对双菌株共转化系统进行改良:目的基因载体携带Pi9抗稻瘟病基因;标记基因载体用潮霉素磷酸转移酶(HPT)作为植物转化选择标记,用GFP作为负选择标记,筛除标记基因分离植株。两种载体的农杆菌转化株混合,分别与水稻品种‘浙恢414’、‘浙粳22’、‘浙11B’、‘日本晴’、‘空育131’和‘粤泰B’的愈伤组织共培养,然后从5%~38.3%的起始愈伤组织筛选获得了转化愈伤组织(HPT+GFP+)。对T0植株进行Pi9基因PCR检测,11.8%~77.8%的T0植株为共转化植株(HPT+GFP+Pi9+)。对共转化植株T1代进行绿色荧光检测,筛选阴性植株(GFP-),再通过PCR筛选Pi9+植株。根据13个T1群体的研究结果,61%的共转化植株在T1代分离出无选择标记转基因植株(HPT-GFP-Pi9+)。转Pi9的无选择标记植株和后代株系对水稻稻瘟病呈抗病反应。因此,本研究通过GFP标记提高了双菌株共转化系统的选择效率,转Pi9的无选择标记水稻株系为水稻抗稻瘟病育种提供了有用的遗传资源。

Generation of Marker-Free Transgenic Rice Carrying the Blast Resistance Gene Pi9 Using Co-transformation Method

Rice blast is a disastrous rice disease caused by Magnaporthe oryzae. Breeding resistant rice cultivars is one of the most economical and effective measures to control rice blast. The rice blast resistance gene Pi9, from the wild rice Oryza minuta, has been cloned and used in tra nsgenic breeding. In order to increase the selection efficiency of marker-free transgenic plants, green fluorescent protein(GFP) was used as a visual genetic marker to improve the two Agrobacterium strains-mediated co-transformation system. The target gene vector carried Pi9 blast resistance gene. The marker gene vector carried hygromycin phosphatase(HPT), the plant transformation selection marker, and GFP, the negative selection marker, to sort out marker-gene segregants. The Agrobacterium strains harboring the above-mentioned vectors were mixed and co-cultured with callus of rice varieties ’ZH414’, ’ZJ22’,’Zhe11 B’, ’Nipponbare’, ’Kongyu 131’ and ’Yuetai B’, respectively. Transformed callus(HPT+GFP+) were obtained from 5%~38.3% the starting callus. T0 plants were assayed by PCR using Pi9-specific primers, and co-transformed plants(HPT+GFP+Pi9+) were obtained from 11.8%~77.8% of the T0 plants. The T1 descendants of co-transformed plants were assayed for green fluorescence detection. The GFP negative plants(GFP-) were selected and further assayed for Pi9 by PCR. Based on the results of 13 T1 populations, 61% of the co-transformed plants segregated marker-free Pi9-transgenic plants(HPT-GFP-Pi9+) in T1 generation. The marker-free Pi9-transgenic plants and progeny lines showed resistance to rice blast. Therefore, the two-strain co-transformation system was improved by using GFP markers, and the marker-free Pi9-transgenic rice lines provide useful genetic resources for rice blast resistance breeding.