结球甘蓝春化相关基因VIN3反义植物表达载体构建及转化

Construction and Transformation of Vernalization-related Genes VIN3 Antisense Plant Expression Vector from Cabbage

以pBI121植物表达载体和BoVIN3-1基因片段为基础,构建结球甘蓝春化相关基因VIN3反义植物表达载体。将BoVIN3-1基因片段反向插入355启动子与GUS基因之间的限制性酶切位点XbaⅠ和SmaⅠ中,构建含反义BoVIN3-1基因的工程质粒pBI35S-BoVIN3-1。通过花蕾微量注射法转化结球甘蓝,获得9株转化植株。PCR检测结果表明,其中5株为阳性植株,阳性株率55.6%。经春化处理的转反义基因植株与对照相比,春化一定程度被推迟。半定量RT-PCR检测结果表明,转化植株在进行低温处理后仍有该基因少量表达,并随春化处理时间的延长转录水平升高,在第50天时表达量达到最高。

Taking plant expression vector pBI121 and BoVIN3-1 gene segment as bases,constructed vernalization-related gens VIN3 antisense plant expression vector from cabbage(Brassica oleracea var. captata L.),then inserted the BoVIN3-1 gene segment into XbaⅠand SmaⅠrestriction enzymes site between CaMV355 promoter and GUS gene in a reverted direction,and constructed plasmid pBI35S-BoVIN3-1.Through transferring it to cabbage flower bud by microinjection method,9 cabbages with Kan-resistance were obtained.The result of PCR amplifications proved that the obtained 5 cabbage were positive plant,and the positive plant rate was 55.6%.Compared the transgenic plant after vernalization treatment with the contrast,the transgenic plant vernalization response be delayed at certain extent.The result of RT-PCR amplifications proved that there was a little expression of VIN3 at small degree after low temperature treatment,and its transcription level increased with the prolongation of treatment time.The transcription reaches the peak at 50 days of vernalization.