Map-based Cloning And Functional Analysis Of Dwarf Gene Dt And Necrotic Gene Nec-t In Maize

Maize (Zea mays L.) is an important crop in world. Its yield plays a role in nationaleconomy. The yield and quality are influenced by environmental stress and biological stress.Maize lodging happened frequently, which influenced the yields seriously. The creation andscreening of new maize dwarf genes and functional analysis have important theoretical andpractical significance. Beyond that its growth and development can be reduced by mutationsof biosynthetic and degradative pathways which can cause necrosis or even programmed celldeath. Hence, Cloning necrotic genes and functional analysis have important theoreticalsignificance. In present study we described the phenotypes of dwarf mutant and necroticmutant, candidate genes and functional analysis. The results were as follows:1. The dwarf mutant was a GA-deficient mutant. The plant height of dwarf plants wasonly49.57%of high individuals. The dwarf mutant plant had shortened internodes, normalcorn, small seed and delayed growth period. Cytological observation revealed that theinternodes were shortened because the stretches of cells in dwarf individuals were inhibited.The exogenous bioactive GAs can successfully made the second sheath elongate and reversedthe dwarf phenotype. But the exogenous BR and IAA did not make the second sheathelongated. So, the dwarf mutant was a GA-deficient mutant.2. Determining and cloning analysis of the candidate gene Dt. We mapped the Dt genebetween markers187790-4and c187790-15. The physical distance was about68Kb andcontained three candidate genes：GRMZM2G040359(GDSL-motif lipase/hydrolase familyprotein), GRMZM2G040673(Plant-specific domain TIGR01568family protein) andGRMZM2G016477(BRASSINOSTEROID INSENSITIVE1-associated receptor kinase1precursor). We sequenced the cDNA and DNA of the three genes and found a singlenucleotide mutant in the663bp of GRMZM2G040673. This led to the lysine change intomethionine. It was a solid foundation for cloning the dwarf gene and studying its functions. We also sequenced eight lines and confirmed the specific mutant. The full length of Dt gene is1720base pair, and encoded383amino acid. The Dt protein belong to OVATE FamilyProtein.3. Temporal and spatial expression patterns of dwarf gene Dt. Dt was specificallyexpressed in stems, tassels and ears by semi-quantitative RT-PCR and quantitative PCR. Theexpression in mutant had no difference with P11. The in situ hybridization of Dt indicated thetranscripts were detected in all meristematic tissue. But expression of Dt in apical meristemwas highest. There was no significant difference between dwarf mutant and P11.4. Functional verification of dwarf gene Dt. OVATE gene was a transcription repressor inplant. Dt-GFP sub-cellular localization analysis indicated that Dt gene was localized in thenucleus. In order to analysis the function of Dt, we constructed Dt-RNAi interfere vector,overexpression vector, and complementary vector. There were a few of dwarf plants intransgenic offspring after Dt was interfered. We also acquired the T0transgenic seedlings ofoverexpression vector and complementary vector. The expression level of ZmKS which wasthe key enzyme of the GA biosynthesis pathway was down regulated. We inferred ZmKS wasthe target of Dt gene.5. Identification of yellow and necrotic mutant gene nec-t. The nec-t mutant plant hadyellow leaves with necrotic spot and reduced level of chlorophyll content at the two-leaf stage,and later on etiolated seedlings died under normal growth conditions. In the mutant leaves,transmission electron microscopy revealed scattered thylakoids, and reduced number of thegrana lamellae and chloroplasts per cell. The nec-t was mapped to a131.7kb region and fourpredicted genes were identified as candidates in the target region. Cloning and sequencing ofthe target region showed that the same7bp insertion mutation occurred in the5′-untranslatedregion (UTR) of the locus LOC100192977. RT-PCR analysis indicated that the nec-ttranscriptional level was mainly expressed in leaves and less in roots and stems both in B73and nec-t mutant. And transcriptional level was significantly reduced in the leaves of mutant.Methionine aminopeptidase that removes the initiator methionine and exposes the penultimateN-terminal amino acid residue plays important role in determining the chloroplast proteinstability. Further studies on functional confirmation of nec-t and the mechanism of lesionformation are currently underway.