Improved nutritive quality and salt resistance in transgenic maize by simultaneously overexpression of a natural lysine-rich protein gene, SBgLR, and an ERF transcription factor gene, TSRF1

Abstract

Maize (Zea mays L.), as one of the most important crops in the world, is deficient in lysine and tryptophan. Environmental conditions greatly impact plant growth, development and productivity. In this study, we used particle bombardment mediated co-transformation to obtain marker-free transgenic maize inbred X178 lines harboring a lysine-rich protein gene SBgLR from potato and an ethylene responsive factor (ERF) transcription factor gene, TSRF1, from tomato. Both of the target genes were successfully expressed and showed various expression levels in different transgenic lines. Analysis showed that the protein and lysine content in T1 transgenic maize seeds increased significantly. Compared to non-transformed maize, the protein and lysine content increased by 7.7% to 24.38% and 8.70% to 30.43%, respectively. Moreover, transgenic maize exhibited more tolerance to salt stress. When treated with 200 mM NaCl for 48 h, both non-transformed and transgenic plant leaves displayed wilting and losing green symptoms and dramatic increase of the free proline contents. However, the degree of control seedlings was much more serious than that of transgenic lines and much more increases of the free proline contents in the transgenic lines than that in the control seedlings were observed. Meanwhile, lower extent decreases of the chlorophyll contents were detected in the transgenic seedlings. Quantitative RT-PCR was performed to analyze the expression of ten stress-related genes, including stress responsive transcription factor genes, ZmMYB59 and ZmMYC1, proline synthesis related genes, ZmP5CS1 and ZmP5CS2, photosynthesis-related genes, ZmELIP, ZmPSI-N, ZmOEE, Zmrbcs and ZmPLAS, and one ABA biosynthesis related gene, ZmSDR. The results showed that with the exception of ZmP5CS1 and ZmP5CS2 in line 9-10 and 19-11, ZmMYC1 in line 19-11 and ZmSDR in line 19-11, the expression of other stress-related genes were inhibited in transgenic lines under normal conditions. After salt treatment, the expressions of the ten stress-related genes were significantly induced in both wild-type (WT) and transgenic lines. However, compared to WT, the increases of ZmP5CS1 in all these three transgenic lines and ZmP5CS2 in line 9-10 were less than WT plants. This study provides an effective approach of maize genetic engineering for improved nutritive quality and salt tolerance.

Figure 1

Comparison of the putative motifs of TSRF1 and its homologs in maize. Motifs of TSRF1 were marked by black lines on the top of the sequences.

Figure 2

Expression of transgenes in T₁ maize (partial results are shown). (A) Semi-quantification RT-PCR analysis of SBgLR in transgenic maize immature seeds (22 DAP); (B) semi-quantification RT-PCR analysis of TSRF1 in transgenic maize leaves; (C) Western blot analysis of SBgLR in transgenic maize immature seeds (20 DAP). M, protein marker; U, non-transformed maize plants. Numbers on the top of each figure represent the transgenic line number.

Figure 3

Segregation of target genes and selectable marker gene in T₂ transgenic plants by DNA dot blot analysis. Two segregated transgenic lines are highlighted in red for 15-9-1 and yellow for 17-8-3, respectively. Transgenic lines that have only Hpt are not indicated. For SBgLR (left), A1 represents positive control and A6 represents negative control. For Hpt (right), D6 represents positive control and D5 represents negative control.

Figure 4

Protein and lysine levels were simultaneously increased in T₁ transgenic seeds. (A) Protein content; (B) lysine content. The crude protein and lysine contents are expressed as g/100 g dry seed. Data are averages of triplicates ± standard deviations. Asterisks, **, denote transgenic lines statistically different from Control by Student t-test at p < 0.01. The lysine content of line 19–11 was not measured due to limited amount of seeds.

Figure 5

TSRF1 improved salt tolerance in maize. (A) Salt tolerance of TSRF1 transgenic maize. Before, control experiment without drought treatment; after, maize seedlings at four-leaf stage were subjected to 200 mM NaCl stress for 48 h; (B) proline and chlorophyll content in the control and transgenic maize. Data are averages of triplicate ± standard deviations. Asterisks, * and **, denote statistically different from wild-type by Student t-test at p < 0.05 and p < 0.01, respectively; (C) relative expression of stress-related genes in wild-type and TSRF1 transgenic maize seedlings before salt treatment; (D) relative expression of stress-related genes in wild-type and TSRF1 transgenic maize seedlings after salt treatment. Total RNA extracted from leaves of maize seedlings before and after 200 mM NaCl treatment for 48 h was used, respectively. Tubulin was used as a reference gene. Data are averages of triplicate ± standard deviations.