Identification of Two Amylases for Construction of Pollen‐Inactivation System in Rice (<scp><i>Oryza sativa</i></scp> L.)

ABSTRACT A critical requirement for exploiting heterosis is the production of an adequate number of male‐sterile female plants that will be further used for production of hybrid F1 seeds by outcrossing with the fertile male parent. Seed production technology (SPT) is a recently developed technology that enables mass production of male‐sterile female parents. SPT is constructed by transformation of a recessive nuclear male‐sterile mutant with a transgenic cassette consisting of three functional modules: a wild‐type gene to restore the fertility of the male‐sterile mutant, a pollen‐killer gene to destroy the pollen grains carrying the transgene and a seed marker gene to distinguish the transgenic seeds from the non‐transgenic seeds. Previous research demonstrated that the maize α‐amylase gene ( ZM‐AA1 ), when linked to the maize polygalacturonase gene ( PG47 ) promoter and brittle‐1 gene ( BT1 ) signal peptide, can effectively disrupt pollen fertility by preventing the starch granule formation in pollen grains. To identify additional amylase genes that can be used for construction of the SPT system in rice, we conducted a BLAST search for amylase genes in rice and maize and tested three candidates: the rice amylase gene OsAA1 , OsAA2 and the maize amylase gene ZM‐AA2 . We found that OsAA1 and ZM‐AA2 , when linked to the PG47 promoter and BT1 signal peptide, were capable of destroying transgenic pollen grains, while OsAA2 lacked this function. Bioinformatics analyses, including protein domain, sequence conservation and phylogenetic studies, revealed that OsAA1 and ZM‐AA2 share structural similarity with ZM‐AA1 whereas OsAA2 exhibited significant differences from ZM‐AA1 in structure and sequence. OsAA1 and ZM‐AA2 identified in this study provide valuable tools for genetic engineering of rice male sterility systems for hybrid rice breeding and production.