Development of transgenic male-sterile rice by using anther-specific promoters identified by comprehensive screening of the gene expression profile database 'RiceXPro'

Abstract

Because genomic selection is designed for the population breeding of allogamous species, a successive outcrossing system is required for efficient use of genomic selection in autogamous crops, such as Oryza sativa L. (rice). Transgenic and dominant male-sterility is a suitable tool for efficient outcrossing of autogamous crops. Though there have been some reports of dominant male-sterile rice developed using transgenic technology, the flowering habit was substandard. Here, to isolate promoters that, when linked to a lethal gene, induce dominant male-sterility while retaining a good flowering habit, we identified 38 candidate genes with anther-specific expression by using the 'RiceXPro' database. We then evaluated the abilities of the near-upstream regions of these genes to induce male-sterility when linked to the lethal gene barnase and introduced into the rice cultivar 'Nipponbare'. Seven of the 38 promoters induced clear dominant male-sterility; promoters expressed in the later stage of anther development induced male-sterility while retaining better flowering habits when compared to ones expressed in the early stage. These seven promoters could potentially be used to facilitate development of an efficient outcross-based breeding system in rice.

Keywords: RiceXPro; anther-specific promoter; barnase; flowering habits; male-sterility; outcrossing; rice (Oryza sativa L.).

Fig. 1

Overview of screening for anther-specific promoters. Screen list of genes showing very high levels of expression in anthers relative to pistils in ‘RiceXPro’ (Sato et al. 2011) for candidates with no or extremely low expression levels in other tissues (e.g., gene in left panel, not right panel). Check the genome information with ‘Rice TOGO Browser’ (Nagamura et al. 2011) and ‘RAP-DB’ (Sakai et al. 2013) to screen for candidates with 1) a large gap from upstream genes. 2) not many restriction enzyme sites or GC-rich repeat regions in the upstream sequences.

Fig. 2

Binary vector used in this study, pZH2Bi-KXB. ASP, anther-specific expressed gene promoter region; aadA, spectinomycin resistance protein; P-35S, CaMV 35S promoter; mHPT, modified hygromycin phosphotransferase; T-nos, nopaline synthase terminator; DT, 35S and nos double terminator; LB, T-DNA left border; RB, T-DNA right border.

Fig. 3

Spikelets (left) and pollen grains (stained with Alexander’s solution; right) of Nipponbare (wild type) and transformants. Left panels: Single underlines indicate the representative constructs that typically generate pollen-producing sterile transformants. Double underlines indicate the representative constructs that typically generate pollen-less sterile transformants. Right panels: blue and red staining indicates non-active and active pollen, respectively.

Fig. 4

Flowering rates of Nipponbare (wild type) and male-sterile transformants. The survey items are illustrated in Supplemental Fig. 2.

Fig. 5

Number of days between heading and flowering.