茎尖法转基因棉花植株真实性鉴定方法探究

棉花茎尖转化法具备不受基因型限制、转化周期短的优点，是理想的棉花转化体系，但据报道其所获得的转基因植株普遍存在遗传不稳定、高代植株基因丢失的现象。以陆地棉TM?1品种为受体材料，利用茎尖转化法将DsRed2载体转入棉花，经卡那霉素筛选获得16株抗性植株，进一步PCR扩增靶基因，获得6株DsRed2基因片段PCR检测为阳性的植株，初步判断该6株为茎尖转化法获得的转基因植株，但经紫外照射，6个转基因植株均未检测到红色荧光。对其进行靶基因RT?PCR，发现DsRed2基因在6个转基因植株中仅有极低量的表达或无表达。进一步对DsRed2载体的非T?DNA片段，即载体骨架部分进行PCR以及植株内生菌培养检测，结果表明，6个转基因植株均含有完整的DsRed2载体，植株可培养出含有完整载体的内生菌，且内生菌经农杆菌16S核糖体RNA（16S rRNA）片段PCR检测结果为阳性，推测由于茎尖侵染形成农杆菌与植株共生关系，造成假阳性株的现象，进而导致高代转基因植株基因丢失、遗传不稳定的现象。旨在建立一套完整的茎尖法转基因棉花植株真实性的鉴定方法，为进一步深入研究提供参考依据。

Exploring of Authenticity Identification Method of Transgenic Cotton Plant by Shoot Tip

The cotton shoot tip transformation method has the advantages of not being restricted by genotype and short transformation cycle， and is an ideal cotton transformation system. However， it is reported that the high?generation plants are generally genetically unstable and the genes are lost in the transgenic plants. In this experiment， the upland cotton TM?1 variety was used as the recipient material， the DsRed2 vector was transferred into cotton by the shoot tip transformation， and 16 resistant plants were obtained by kanamycin screening. The target gene was further amplified by PCR and 6 plants with DsRed2 gene fragment PCR test positive were obtained. The 6 plants were preliminarily judged to be transgenic plants obtained by the shoot tip transformation， but after UV irradiation， no red fluorescence was detected in the 6 transgenic plants. The target gene RT?PCR was performed and found that the DsRed2 gene had only a very low expression or no expression in the 6 transgenic plants. The non?T?DNA fragments of the DsRed2 vector， that is， the backbone of the vector， were subjected to PCR and plant endophyte culture testing. The results showed that the 6 transgenic plants all contained the complete DsRed2 vector and the plant could cultivate endophytes containing the complete vector. The endophyte was positive by PCR of the 16S rRNA fragment of Agrobacterium. It is speculated that the symbiotic relationship between Agrobacterium and plants due to shoop tip infection， which in turn leading to the phenomenon of false positive plants， and the loss of genes and genetic instability of high?generation transgenic plants. The purpose of this research is to establish a complete set of methods for identifying the authenticity of transgenic cotton plants by the shoot tip method， and to provide reference for further in?depth research.