对转蚕丝芯蛋白轻链基因棉花的分析

利用根癌农杆菌介导转化法，将棉纤维特异表达启动子GAE6-3A驱动的蚕丝芯蛋白轻链基因（FBN）转入陆地棉R15中。T₀代转基因再生株FNB基因PCR检测阳性率达70%。随机选取4个转基因株系T₁代材料的Southern杂交显示，3个为双拷贝插入、1个为单拷贝插入；Northern分析结果证实FBN基因在转基因棉纤维中表达。转基因后代的纯合选育主要以田间卡那霉素检测结合实验室内GUS组织化学检测进行，其中4个株系已获得了T₃代转基因材料，其他若干个转化子也获得T₁代和T₂代的不同材料。对6个转基因棉花株系后代纤维检测结果表明，蚕丝芯蛋白轻链基因对棉花纤维品质的影响主要体现于对纤维强度的改良。H18、H32、H34株系转基因棉花后代纤维强度较对照显著提高，其中H18纤维强度提高12.3%。

Analysis of Cotton (Gossypium hirsutum L.) Plants Transformed with a Silkworm Fibroin Light Chain Gene

By Agrobacterium-mediated transformation,a silkworm fibroin light chain gene(FBN),driven by a fiber-specific promoter GAE6-3A,was introduced into the upland cotton G.hirsutum L.cv R15.The plant expression vector used in this study also contains a GUS gene,and a npt II gene driven by 35S promoter,respectively.Kanamycin-resistance analysis,GUS-histochemical staining,and PCR detection were conducted in the 20 T0 generation plants form 11 different transgenic lines.Seventeen plants showed kanamycin resistance,13 plants were positive in GUS detection,and 14 were positive by PCR analysis for the silkworm fibroin light chain gene FBN.Thirteen positive plants were obtained by the three examination methods among the 20 T0 generation plants.Southern hybridization of T1 progenies of lines H18,H21,H32,and H34 were performed,the result showed that among the four T1 lines,three lines contained two copies and one line contained a single copy of the transgene,no clear hybridization signal was detected for non-transformed control plants,indicating that FBN gene was inserted into the cotton genome by Agrobacterium-medicated transformation.Northern analysis demonstrated that the FBN gene was indeed expressed in the transgenic cotton fiber of above four transgenic lines.Kanamycin-resistance assay and GUS-histochemical staining was used for screening transgenic progenies.Kanamycin assay was performed in the field as a preliminary screening,which may be influenced by climate and other circumstance factors,however,the operation is simple and fast,laying the basis for further selection by GUS histochemical localization.Proceeded the two methods together,the limitation of each measure can be compensated,accordingly the efficiency of homozygous breeding of transgenic progenies could be increased obviously.Till now four T3 positive lines,in addition to several T1 or T2 transgenic plants were obtained,and the foreign gene could be inherited stably form generation to generation.Analysis of fiber quality traits showed that,among the six transgenic lines whose cotton fiber quality was evaluated,the fiber strength of three lines were increased,with the highest in transgenic line H18. These results demonstrate that plant genetic engineering of silkworm fibroin genes has a great potential for improving cotton fiber quality.