转As6G-FFT基因烟草的阳性鉴定及基因拷贝数测定

Identification and Copy Number of As6G-FFT in Transgenic Tobacco Plant

【目的】为验证转As6G-FFT烟草的基因功能,筛选稳定遗传的阳性株系材料,以建立基于SYBR Green的实时荧光定量PCR的转基因拷贝数检测方法。【方法】利用PCR检测、实时荧光定量PCR(Real-time quantitative PCR,qRT-PCR)技术及生理指标分析鉴定转As6G-FFT基因阳性烟草植株,并利用基于SYBR Green的实时荧光定量PCR鉴定阳性转基因烟草中As6G-FFT基因的拷贝数。【结果】(1)基于PCR检测,14个转基因烟草叶片均能扩增出目的片段,表明14个株系中均已成功转入目的基因As6G-FFT;(2)14个转基因株系中As6G-FFT基因表达量呈极显著(P<0.01)或极其显著上升(P<0.001),其中6个株系的表达量呈极其显著升高(P<0.001);且其表达量与野生型相比最高提高215.13倍;(3)基于生理指标,测定转As6G-FFT基因烟草的果聚糖含量,发现14个转基因株系中果聚糖含量呈极显著(P<0.01)或极其显著上升(P<0.001),其中13个株系的果聚糖含量极其显著升高(P<0.001);且其果聚糖含量与野生型相比最高提高10.47倍;(4)基于SYBR Green实时荧光定量PCR构建As6G-FFT和Nt ACT基因的标准曲线,分别为y=-0.2907x+3.0145和y=-0.2813x+8.0141,R^(2)均为1;在检测的14个转基因株系中As6G-FFT基因拷贝数为1~3,其中1、2和3拷贝的单株数分别占总数的35.7%、50.0%和14.3%。【结论】本研究从DNA、RNA和生理水平综合进行阳性转基因烟草的鉴定,鉴定结果更为准确。此外,还建立了基于SYBR Green实时荧光定量PCR的转基因烟草中外源As6G-FFT基因拷贝数检测方法,可用于快速、高效地估算转基因烟草中外源基因拷贝数,为后续获得稳定遗传材料提供筛选依据。

【Objective】Functions,identification,and copy number of As6G-FFT in the transgenic tobacco plants were studied.【Method】PCR,qRT-PCR,and physiological analysis were performed to confirm the transgenic tobacco plants being As6G-FFT-positive and elucidate the functions of the gene.SYBR green-based qRT-PCR was applied to determine the copy number of the gene in the transgenic plant.【Result】(1)The target fragment was amplified on the leaves of 14 tobacco plants by PCR assuring a successful transfer of As6G-FFT.(2)In varying degrees,the gene expressions in the 14 transgenic lines were higher than in the wild-type.Six of the lines were extremely significantly higher than the wild-type counterpart,with an accumulation topped 215.13-fold.(3)The fructan contents were higher in the leaves of the transgenic than the wild-type plants.Thirteen of the transgenic lines contained extremely significantly more fructan than the wild-type with the highest accumulation of 10.47-fold.(4)With correlation coefficients of 1,the SYBR green-based qRT-PCR standard curves of y=-0.2907x+3.0145 was obtained for As6G-FFT and y=-0.2813x+8.0141 for NtACT.Of the 14 transgenic lines,35.7%contained only one of the gene,50.0%had 2,and 14.3%3 copies.【Conclusion】Transgenic tobacco plants with As6G-FFT were identified based on the DNA,RNA,and physiological aspects.The SYBR green-based qRT-PCR method rapidly and efficiently determined the number of exogenous As6G-FFT transferred into the plants and could be a convenient tool for screening and acquisition of stable genetic materials.