一种可视化筛选转基因通用载体pCamRUBY的构建和应用研究

doi:10.13560/j.cnki.biotech.bull.1985.2024-1133

摘要/Abstract

摘要：

目的构建并验证一种可视化筛选转基因通用载体pCamRUBY，以期提高植物遗传转化效率。方法在以RUBY作为报告基因的基础上利用2A技术构建成一个通用表达载体pCamRUBY，以Spe和Kan抗性基因分别构建验证载体pCamSpeRUBY和pCamKanRUBY，并分别以拟南芥和棉花为材料进行遗传转化验证和色素含量鉴定。结果该载体具有可视化程度高，载体序列小，内含由Sal I、Apa I、Xba I、Sac I和Spe I组成的MCS位点和T2A（optimized）序列等特点。分别获得可裸眼观察筛选的具有红色表型性状的转基因拟南芥和棉花阳性植株或组织，并可得到稳定遗传后代。结论该通用表达载体pCamRUBY可直接连接外源基因正常表达、可稳定遗传且可直接裸眼观察筛选的红色性状转基因阳性植株，且不影响包含叶绿素含量在内的其他表型性状。

关键词: 遗传转化, 通用载体, 报告基因, RUBY

Abstract:

Objective To construct and verify an universal vector for visual screening transgenic plants and improving the efficiency of plant genetic transformation. Method Having gene RUBY as the reporter gene, a universal expression vector pCamRUBY was constructed using 2A technology. Validation vectors pCamSpeRUBY and pCamKanRUBY were constructed using Spe and Kan resistance genes, respectively. Arabidopsis and cotton were used as the test materials for genetic transformation and pigment content identification. Result This vector has the characteristics of high visualization, small vector sequence, MCS sites composed of Sal I, Apa I, Xba I, Sac Iand Spe I, and T2A (optimized) sequences. The red positive transgenic Arabidopsis and cotton plats and tissues were visually screened with the naked eye respectively, and stable genetic inheritance was obtained. Conclusion The universal expression vector pCamRUBY can directly be linked to the normal exogenous genes, and the positive transgenic plants show red character. The transgenic plants can be directly observed and screened with naked eye, and not affect other phenotypic traits including chlorophyll content of positive plants.

Key words: genetic transformation, universal vector, reporter gene, RUBY

豆硕, 丁若羲, 孙星, 郭文静, 孔文慧, 袁静贤, 张冬梅, 王省芬, 马峙英, 吴金华, 吴立柱. 一种可视化筛选转基因通用载体pCamRUBY的构建和应用研究[J]. 生物技术通报, 2025, 41(12): 66-73.

DOU Shuo, DING Ruo-xi, SUN Xing, GUO Wen-jing, KONG Wen-hui, YUAN Jing-xian, ZHANG Dong-mei, WANG Xing-fen, MA Zhi-ying, WU jin-hua, WU Li-zhu. Construction and Application Study of a General Vector pCamRUBY for Visual Screening of Transgenes[J]. Biotechnology Bulletin, 2025, 41(12): 66-73.

图/表 7

表1 引物名称及序列信息

Table 1 Primers' names and sequence information

引物名称 Primer name	引物序列 Primer sequence （5′-3′）
RUBYF	CCGTGCACATCAACGACACC
RUBYR	GGAGGTGAACTTGTAGGAGC
AtActinF	CTGGTGATGGTGTGTCTCACAC
AtActinR	GCGATCCAGACACTGTACTTCC
GhActinF	GATCCTTCCTGATATCCACATCG
GhActinR	GGCACACTGGTGTTATGGTTGGG

图1 载体结果示意图和测序比对结果A：pCamRUBY载体图谱；B：pCamSpeRUBY载体图谱和测序比对结果；C：pCamKanRUBY载体图谱和测序比对结果

Fig. 1 Schematic diagram of vector results and sequencing alignment resultsA: pCamRUBY vector profile. B: pCamSpeRUBY vector profile and sequencing alignment results. C: pCamKanRUBY vector profile and sequencing alignment results

图2 转基因的拟南芥的筛选A：利用Spe抗性筛选获得的红色拟南芥植株；B：营养土直接播种目测筛选的红色性状拟南芥植株；C：T2代pCamSpeRUBY转基因拟南芥植株；箭头所示为转基因阳性植株；标尺为1 cm

Fig. 2 Transgenic Arabidopsisthaliana screeningA: Red Arabidopsis were obtained through Spe resistance screening. B: Red Arabidopsis were directly screened with naked eye on nutrient soil. C: T2 generation pCamSpeRUBY transgenic Arabidopsis. The arrow indicates the transgenic positive plants. Bar=1 cm

图3 转基因拟南芥表型观察A：野生型和转基因拟南芥花序表型观察；B：野生型和转基因拟南芥抽薹期表型观察；C：野生型和转基因拟南芥（T3代）成苗单株表型观察；D：野生型和转基因拟南芥种子表型观察；E：转基因拟南芥RUBY基因的PCR电泳结果；F：转基因拟南芥AtActin基因的PCR电泳结果。M：DNA maker DL2000；1-2：抗性筛选获得的转基因拟南芥植株；3-5：直接播种获得的转基因植株；P：质粒pCamRUBY；WT：野生型对照拟南芥植株；c：空白对照；标尺为1 cm

Fig. 3 Phenotype of transgenic A. thalianaA: Phenotype of wild type and transgenic Arabidopsis inflorescence. B: Phenotype of bolting wild type and transgenic Arabidopsis. C: Phenotype of wild type and transgenic Arabidopsis plants. D: Phenotype of wild type and transgenic Arabidopsis seeds. E: PCR identification of RUBY gene in transgenic Arabidopsis. F: PCR identification of AtActin gene in in transgenic Arabidopsis. M: DNA maker DL2000. 1-2: Transgenic Arabidopsis plants obtained through resistance screening. 3-5: Transgenic Arabidopsis plants obtained by direct seeding. P: Plasmid pCamRUBY. WT: Wild-type control Arabidopsis plant. c: Blank control. Bar=1 cm

表2 转基因拟南芥种子分离比统计

Table 2 Separation ratio statistics of transgenic Arabidopsis seeds

荚果编号 No. of pod	种子数 Number of seeds	红色种子数 Number of red seeds	黄褐色种子数 Number of tawny seeds
1	28	21	7
2	22	17	5
3	23	18	5
4	32	25	7
5	20	15	5
6	19	15	4
7	25	18	7
8	29	21	8
9	24	18	6
10	26	19	7
Total	248	187	61

图4 棉花遗传转化愈伤和植株表型观察和阳性植株PCR鉴定A：转基因棉花的愈伤组织呈现红色性状；B：转基因棉花阳性植株幼苗呈现红色性状；C：转基因棉花阳性植株成苗呈现红色性状；D：野生型和T1代转基因棉花表型观察；E：转基因棉花RUBY基因的PCR电泳结果；F：转基因棉花GhActin基因的PCR电泳结果。M：DNA maker DL2000；1-3：T0代转基因棉花植株；P：质粒pCamRUBY；WT：野生型对照棉花植株；c：空白对照；标尺为1 cm

Fig. 4 Phenotypic observation of genetically transformed cotton calli and plants and PCR identifications of positive plantsA: Red transgenic cotton callus. B: Red transgenic cotton seedlings. C: Red transgenic cotton plants. D: Phenotype of wild type and T1 transgenic plants. E: PCR identification of RUBY gene in transgenic cotton. F: PCR identification of GhActin gene in transgenic cotton. M: DNA marker DL2000; 1-3: T0 transgenic cotton plants; 4-6: T1 transgenic cotton plants; P: plasmid pCamRUBY; WT: wild-type cotton J668; c: blank control; Bar=1 cm

图5 转基因棉花和拟南芥的叶绿素和甜菜红素的含量检测A：转基因棉花叶绿素含量测定；B：转基因拟南芥叶绿素含量测定；C：转基因植株甜菜红素含量测定

Fig. 5 Content detection of chlorophyll and betacyanin in transgenic cotton and ArabidopsisA: Determination of chlorophyll content in transgenic cotton. B: Determination of chlorophyll content in transgenic A. thaliana. C: Determination of betacyanin content in transgenic plants. *P<0.05; ***P<0.001

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