title: Studies on gene structure, enzymatic activity and regulatory mechanism of acetohydroxy acid isomeroreductase from G2 pea.
abstract: TheAAIR genomic DNA of G2 pea (Pisum sativum L.) was amplified by PCR method. Sequence analysis showed that it was composed of 8 introns and 9 exons with three of the introns containing specific A/T-rich endogenous promoter regions. Molecular hybridization experiments revealed that the expression of AAIR remained at a high level before and after flowering if grown in short day growth chambers. However, when grown under long day conditions, the level of AAIR expression declined very rapidly after flowering. This variation of AAIR expression is consistent with the change of enzymatic activity of acetohydroxy acid isomeroreductase. Functional complementation experiments carried out using an acetohydroxy acid isomeroreductase deficientE. coli strain showed that these cells could not grow on M9 medium without addition of branched-chain amino acids unless they were transformed with theAAIR expression vector. Further study revealed that overexpression of the peaAAIR cDNA in acetohydroxy acid isomeroreductase deficientE. coli strain enhanced significantly its branched-chain amino acid biosynthetic capacity. Results from gel shift experiments showed that fractions of pea nuclear protein extracts could bind specifically to some A/T rich regions present in introns of theAAIR gene. The A/T-rich-region-binding proteins remained at a steady level in the non-senescing apical buds of short-day grown G2 pea. In the rapid-senescing apical buds of long-day grown G2 pea, the levels of these proteins declined rapidly after flower initiation. Therefore, the nuclear protein binding capacities to endogenous promoter regions may constitute an important mechanism to regulateAAIR gene expression.
authors.affiliation: The National Laboratory of Protein Engineering and Plant Genetic Engineering, Peking University, 100871, Beijing, China.
authors.forename: Yunjian
authors.initials: Y
authors.lastname: Xu
authors.forename: Xuesong
authors.initials: X
authors.lastname: Gu
authors.forename: Jun
authors.initials: J
authors.lastname: Li
authors.forename: Qing
authors.initials: Q
authors.lastname: Li
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Davies
authors.forename: Yuxian
authors.initials: Y
authors.lastname: Zhu