Construction and Application Study of a General Vector pCamRUBY for Visual Screening of Transgenes

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

Abstract

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

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.

Figures/Tables 7

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

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

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

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

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

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

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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