GUS基因和NPTII基因表达的相关性及其在转基因棉花检测研究中的应用

Expression of GUS Gene and NPTII Gene and Their Application in the Detecting of Transgenic Cotton

利用农杆菌介导转化法,将含有35S启动子驱动NPTII基因和GUS基因以及棉纤维特异表达启动子E6驱动目的基因FB的植物表达载体转入到常规棉花R15中。重点分析了GUS基因和NPTII基因在愈伤诱导阶段、T0代及T1代转基因棉花中的表达情况。综合两个基因的表达来进行转基因棉花的阳性鉴定,可以为转基因棉花后代的纯合选育提供双重保障。7个转基因株系选育到T3代共获得株行51个,卡那霉素检测多数株行阳性率在90%以上,其中21个株行阳性率达100%。

By Agrobacterium-mediated method, the plant expression vector contained the target gene FB driven by a cotton fiber specific expression promoter E6 was transferred into cotton cultivar R15. The vector also contains a GUS gene,and a NPTII gene driven by 35S promoter, respectively. The expression of GUS gene and NPTII gene was analyzed in the stage of resistant calli selected, To and T1 generation transgenic plants. These two genes are in the same T-DNA in the vector, their expression has high relativity. Because the phenomena of transgenic silencing and the effect of insert sites, the expression of GUS gene and NPTII gene has some differences. We obtained the resistant calli through detecting the expression of GUS gene and NPTII gene together. Kanamycin resistance, GUS staining, and PCR detection were conducted on the 15 putative To generation transgenic plants. Among them, 11 plants were positive for the transgene according To PCR detection, the positive ratio reached 73.3 %. 12 plants showed kanamycin resistance, 10 plants were GUS-staining positive, the expression of NPTII gene and GUS gene showed high relativity. 10 positive plants were obtained through the three derected methods among the 15 To generation transgenic plants. The breeding of transgenic progeny also relied on both kanamycin examination and GUS staining. Of T1 transgenic progeny, the expression of GUS gene and NPTII gene has significant difference, so there has some limitation for To as a certain extent for screening the transgenic progeny only by kanamycin examination. Kanamycin assay was performed in the field, climate and other circumstance -factors may influence the result. However, being simple and fast, this assay can employed as a preliminary screen, three times of kanamycin assay were performed during the period of cotton vegetable development, and then GUS staining were acted based on the result of kanamycin resistant test. Proceeded the two methods together , the limitation of each measure can be compensated, accordingly the efficiency of homozygous breeding of transgenic progeny could be increase obviously. Seven T1 generation transgenic lines were all in accordance with Mendelian patterns of inheritance in the ratio of resistant plants To non-resistant plants through kanamycin assay and GUS staining. It is a complicated process that the exogenous gene integrated into cotton genome, and need To screen many generations for obtained homozygous progeny. Collaboration of kanamycin test and GUS staining is a virtual application for transgenic cotton detecting. In the T3 generation transgenic progeny from the seven transformants, 51 transgenic lines had been obtained, the kanamycin resistant ratio of most of these lines were more than 90% ,and 21 transformation lines reached 100% among them,and had been homozygous possibly. The foreigns gene could be inherited stably through resistance screening generation by generation.