Functional Characterization of Soybean Glyma04g39610 as a Brassinosteroid Receptor Gene and Evolutionary Analysis of Soybean Brassinosteroid Receptors

摘要

Brassinosteroids (BR) play important roles in plant growth and development. Although BR receptors have been intensively studied in Arabidopsis , the BR receptors in soybean remain largely unknown. Here, in addition to the known receptor gene Glyma06g15270 ( GmBRI1a ), we identified five putative BR receptor genes in the soybean genome: GmBRI1b , GmBRL1a , GmBRL1b , GmBRL2a , and GmBRL2b . Analysis of their expression patterns by quantitative real-time PCR showed that they are ubiquitously expressed in primary roots, lateral roots, stems, leaves, and hypocotyls. We used rapid amplification of cDNA ends (RACE) to clone GmBRI1b ( Glyma04g39160 ), and found that the predicted amino acid sequence of GmBRI1b showed high similarity to those of AtBRI1 and pea PsBRI1. Structural modeling of the ectodomain also demonstrated similarities between the BR receptors of soybean and Arabidopsis . GFP-fusion experiments verified that GmBRI1b localizes to the cell membrane. We also explored GmBRI1b function in Arabidopsis through complementation experiments. Ectopic over-expression of GmBRI1b in Arabidopsis BR receptor loss-of-function mutant ( bri1-5 bak1-1D ) restored hypocotyl growth in etiolated seedlings; increased the growth of stems, leaves, and siliques in light; and rescued the developmental defects in leaves of the bri1-6 mutant, and complemented the responses of BR biosynthesis-related genes in the bri1-5 bak1-D mutant grown in light. Bioinformatics analysis demonstrated that the six BR receptor genes in soybean resulted from three gene duplication events during evolution. Phylogenetic analysis classified the BR receptors in dicots and monocots into three subclades. Estimation of the synonymous ( K s ) and the nonsynonymous substitution rate ( K a ) and selection pressure ( K a / K s ) revealed that the K a / K s of BR receptor genes from dicots and monocots were less than 1.0, indicating that BR receptor genes in plants experienced purifying selection during evolution.