Map-based Cloning And Functional Analysis Of The Dwarf Gene D2003in Maize (Zea Mays L.)

Appropriate plant height is one of the most important agronomic traits in maize breeding programs because it is crucial for planting density and lodging resistance. Understanding of the genetic mechanism of plant height is extraordinary useful in crop yield improvement. In this study, we isolated a natural mutant d2003from K36inbred background with less than one-third of K36plant height, and possesses more but severe shortened internodes which cell number markedly reduced.By positional cloning we cloned the D2003gene, sequence analysis and allelism test found that it is a allele of Viviparous8(Vp8), which encodes a putative membrane localized glutamate carboxypeptidase, but its mechanism is not well understood. Sequence alignment revealed that a single-base insertion resulting in a premature stop codon in the first exon of d2003. The full-length cDNA fragments of K36was amplified, constructed to pLEELA vector, and transformed into the ampl mutant, a orthologou of d2003, most of the transgenic plants show wild-type phenotype. At the same time, we introgressed the d2003gene into16background using backcrossing strategies combined with molecular marker selection, and all of them show the dwarf phenotype.Expression analysis demonstrated that D2003is mainly expressed in stem apex, young leaves, as well as developing vascular tissue, medium in tassel, and low in ear and root, diffusely through shoot and root. Subcellular localization revealed that D2003/VP8protein is co-localized with the endoplasmic reticulum (ER) marker protein HDEL-GFP, demonstrate that D2003/VP8is a ER localized protein. At the same time, protein sequence analysis found that there are signal-peptide and transmembrane-regions at N-terminal and play important role in correct protein localization and is required for its function.In addition, d2003is sensitive to exogenous GA3, IAA,6-BA and BR application, four phytohormones predominantly promote cell division or elongation, but all of the treatment could not rescue the dwarf phenotype absolutely. Moreover, RNA sequencing detected a protein kinase gene, GRMZM2G035098, which plays a key role in cellular processes, including cell division, cell proliferation, as well as proper cell cycle progression and chromosome maintenance.Thus, we propose that D2003may participate in regulating internode elongation via participating in cell division during plant height development in maize.