Overexpression of ZmIPT2 gene delays leaf senescence and improves grain yield in maize

Abstract

Cytokinins (CTKs) are a major phytohormone group that are significant in the promotion of cellular division, growth, and divergence. Isopentenyl transferase (IPT) regulates a rate-limiting step in plant CTK synthesis, promotes the synthesis of isopentenyl adenonucleotides from 5-AMP and isopentenyl pyrophosphate, and then converts both these chemicals into various CTKs. Here, the full-length cDNA of ZmIPT2, which encodes 322 amino acids, was isolated and was introduced into a maize inbred line by Agrobacterium-mediated transformation. In both controlled environments and field experiments, the overexpression of ZmIPT2 gene in the transformed plants delayed leaf senescence. Compared to the receptor line, the transgenic maize lines retained higher chlorophyll levels, photosynthetic rates, and cytokinin content for an extended period of time, and produced significantly higher grain yield by a margin of 17.71-20.29% under normal field planting conditions. Subsequently, ten possible genes that interacted with ZmIPT2 were analyzed by qRT-PCR, showing that the expression pattern of GRMZM2G022904 was consistent with ZmIPT2 expression. Through comprehensive analysis, we screened for transgenic lines with stable inheritance of ZmIPT2 gene, clear functional efficiency, and significant yield improvement, in order to provide theoretical basis and material support for the breeding of new high-yield transgenic maize varieties.

Keywords: ZmIPT2 gene; grain yield; leaf senescence; maize; overexpression.

FIGURE 1

The characterization of the ZmIPT2 protein. (A) Transmembrane domain prediction of ZmIPT2. (B) Prediction of signal peptide and splice siteS. (C) Hydrophobicity analysis of ZmIPT2 coding protein. (D) Analysis of flanking sequence of ZmIPT2 gene. (E) Secondary structure analysis of the ZmIPT2. The helix (h), extended strand (e), coil(c), and turn (t) are indicated in different color as blue, red, yellow, and green, respectively. (F) 3-D structure model of ZmIPT2.

FIGURE 2

Relative expression levels of transgenic ZmIPT2 gene in maize leaves at different stages in T2 and T3 generation. (A) Shows Relative expression levels of transgenic ZmIPT2 gene in maize leaves at different stages in T2 generation, V6 (sixth leaf), R1 (silking), R2 (blister stage) and R6 (physical maturity); (B) shows Relative expression levels of transgenic ZmIPT2 gene in maize leaves at different stages in T3 generation. The error bars denote standard deviations of the qRT-PCR signals (n = 3). There was significant difference or extremely significant difference in the expression level of ZmIPT2 (**P < 0.01, Student t-test).

FIGURE 3

Detection of physiological and biochemical indexes of transgenic maize lines with ZmIPT2 gene. (A) Shows the relative chlorophyll content of transgenic lines at different growth stages of maize, V6 (sixth leaf), R1 (silking), R2 (blister stage) and R6 (physical maturity); (B) shows the photosynthetic rate content of transgenic lines at different growth stages of maize, V6 (sixth leaf), R1 (silking), R2 (blister stage) and R6 (physical maturity); (C) shows the content of cytotaxon (CTK) of transgenic lines at different growth stages of maize, V6 (sixth leaf), R1 (silking), R2 (blister stage) and R6 (physical maturity). (D) The comparison of leaf greenness in T3 transgenic maize lines with ZmIPT2 gene at generation at mature stage, C01, negative control; 1, DNIPT2-C14; 2, DNIPT2-C33; 3, DNIPT2-C34. There was significant difference or extremely significant difference in the expression level of ZmIPT2 (**P < 0.01, Student t-test).

FIGURE 4

The ear and grain of transgenic maize lines with ZmIPT2 gene and the receptor line C01. (A) The ear of T2 transgenic maize lines with ZmIPT2 gene and the receptor line C01. (B) The ear of T3 transgenic maize lines with ZmIPT2 gene and the receptor line C01. C01, negative control; 1, DNIPT2-C24; 2, DNIPT2-C33; 3, DNIPT2-C34.

FIGURE 5

Gene analysis of ZmIPT2 interaction. (A) The predictive and analysis of ZmIPT2 interaction protein. (B) Relative expression levels of transgenic GRMZM2G022904_P01 gene in maize leaves at different stages. (C) Relative expression levels of transgenic GRMZM2G168681 gene in maize leaves at different stages. (D) Relative expression levels of transgenic GRMZM2G147721 gene in maize leaves at different stages. There was significant difference or extremely significant difference in the expression level (**P < 0.01, Student t-test).