转<i>As</i>6<i>G-FFT</i>基因烟草的阳性鉴定及基因拷贝数测定

铁原毓; 文军琴; 田洁

doi:10.19303/j.issn.1008-0384.2022.005.006

转As6G-FFT基因烟草的阳性鉴定及基因拷贝数测定

doi: 10.19303/j.issn.1008-0384.2022.005.006

铁原毓^1,,
文军琴¹,
田洁^{1, 2, ,}

1.
青海大学农林科学院/青海省蔬菜遗传与生理重点实验室，青海　西宁　810016
2.
省部共建三江源生态与高原农牧业国家重点实验室，青海　西宁　810016

基金项目: 国家自然科学基金项目（31960590）；青海省科技厅重点实验室项目（2020-ZJ-Y02）；中国科学院“西部之光”项目（2019年）

详细信息

作者简介:
铁原毓（1997-），女，硕士研究生，研究方向：蔬菜分子生物学（E-mail：healer2727@163.com）

通讯作者:
田洁（1986-），女，博士，副研究员，研究方向：蔬菜生理与分子生物技术（E-mail：tiantian8092001@163.com）

中图分类号: S 572
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出版历程
- 收稿日期: 2022-01-12
- 录用日期: 2022-01-12
- 修回日期: 2022-03-23
- 网络出版日期: 2022-04-24
- 刊出日期: 2022-05-28

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

TIE Yuanyu^1
,,
WEN Junqin¹,
TIAN Jie^{1, 2
, ,}

1.
Qinghai Key Laboratory of Vegetable Genetics and Physiology/Agriculture and Forestry Sciences Institute, Qinghai University, Xining, Qinghai　810016, China
2.
State Key Laboratory of Plateau Ecology and Agriculture, Xining, Qinghai　810016, China

摘要

摘要: 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NtACT基因的标准曲线，分别为y=−0.290 7x+3.014 5和y=−0.2813x+8.0141，R²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。
- As6G-FFT基因 /
- 转基因烟草 /
- 阳性鉴定 /
- 拷贝数
Abstract: 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.
- As6G-FFT /
- transgenic tobacco /
- positive identification /
- copy number

HTML全文

图 1 转As6G-FFT基因烟草PCR检测

M：DNA分子量标准（2 000 bp）；WT：野生型烟草；N1～N14：转基因烟草株系。下同。

Figure 1. PCR detection of As6G-FFT in transgenic tobacco plants

M: DNA marker (2 000 bp); WT: wild-type tobacco; N1-N14: transgenic tobacco lines. Same for the following.

下载: 全尺寸图片幻灯片

图 2 转As6G-FFT基因烟草的基因表达量

**：差异极显著（P＜0.01）；***：差异极其显著（P＜0.001）。图3同。

Figure 2. As6G-FFT expressions in transgenic and wild-type tobacco plants

**: extremely significant difference (P＜0.01), ***: extremely significant difference (P＜0.001) ; The same as Fig.3.

下载: 全尺寸图片幻灯片

图 3 转As6G-FFT基因烟草的果聚糖含量

Figure 3. Fructan contents in transgenic and wild-type tobacco plants

下载: 全尺寸图片幻灯片

图 4 As6G-FFT和NtACT基因实时荧光定量PCR的标准曲线

A：As6G-FFT基因；B：NtACT基因。图5、6同。

Figure 4. Real-time fluorescent quantitative PCR standard curves of As6G-FFT and NtACT

A: As6G-FFT; B: NtACT. Same for Fig. 5, 6.

下载: 全尺寸图片幻灯片

图 5 As6G-FFT和NtACT基因实时荧光定量PCR的熔解曲线

Figure 5. Real-time fluorescent quantitative PCR melting curves of As6G-FFT and NtACT

下载: 全尺寸图片幻灯片

图 6 As6G-FFT和NtACT基因实时荧光定量PCR的扩增曲线

Figure 6. Real-time fluorescent quantitative PCR amplification curves of As6G-FFT and NtACTs

下载: 全尺寸图片幻灯片

表 1 引物序列

Table 1. Primer sequence

引物 Primer	引物序列（5′-3′） Primer sequence	目的 Purpose
As6G-FFT-clone	F: ATGGATGCTCAAGACATTGAG TC	阳性PCR检测 Positive PCR detection
As6G-FFT-clone	R: TTAAAAATGATAAAAATCATTG TAAGTGGAGTTC	阳性PCR检测 Positive PCR detection
As6G-FFT	F: TGGCTCTTTACGCACTCA	实时荧光定量 PCR分析 qRT-PCR analysis
As6G-FFT	R: TCGCACTCGTCCTACCTC
NtACT	F: AATGATCGGAATGGAAGCTG
NtACT	R: TGGTACCACCACTGAGGACA

下载: 导出CSV

表 2 转基因烟草中As6G-FFT基因拷贝数

Table 2. Copy number of As6G-FFT in transgenic tobacco plants

转基因烟草 Transgenic tobacco	As6G-FFT基因 As6G-FFT gene			NtACT基因 NtACT gene			浓度对数值的比值 Log value ratio of concentration	拷贝数 Copy number
转基因烟草 Transgenic tobacco	C_t	熔解温度 T_m	浓度对数值 Log value of concentration	C_t	熔解温度 T_m	浓度对数值 Log value of concentration	浓度对数值的比值 Log value ratio of concentration	拷贝数 Copy number
N1	19.14	83.81	2.55	20.60	80.13	2.22	1.15	1
N2	20.44	83.67	2.93	23.40	80.12	1.43	2.04	2
N3	20.81	83.70	3.03	20.78	80.10	2.17	1.40	1
N4	21.08	83.79	3.11	20.62	80.25	2.21	1.41	1
N5	21.66	83.73	3.28	23.57	80.23	1.38	2.37	2
N6	20.62	83.60	2.98	21.76	80.04	1.89	1.57	2
N7	19.72	83.61	2.72	21.53	80.23	1.96	1.39	1
N8	18.98	83.78	2.50	21.33	80.38	2.01	1.24	1
N9	23.51	83.75	3.82	23.82	80.19	1.31	2.91	3
N10	22.48	83.66	3.52	22.39	80.08	1.71	2.05	2
N11	22.46	83.79	3.51	21.78	80.21	1.89	1.86	2
N12	21.80	83.84	3.32	22.60	80.29	1.66	2.01	2
N13	22.76	83.69	3.60	22.75	80.12	1.61	2.23	2
N14	22.72	83.66	3.59	23.42	80.03	1.43	2.52	3

下载: 导出CSV

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