A two gene- two promoter system for enhanced expression of a restorer gene (barstar) and development of improved fertility restorer lines for hybrid seed production in crop plants

Abstract

We report in this study, strategies for enhancing and extending tissue-specific expression of a restorer gene (barstar) and their use in the development of improved fertility restorer lines for transgenic male sterile (barnase) lines for hybrid seed production. In the first strategy, a chimeric promoter was developed by combining regulatory elements of two overlapping tapetum-specific promoters (A9 and TA29) by placing a 275bp fragment of the A9 promoter (minus the TATA box) upstream to a functional 330bp fragment of the TA29 promoter. Analysis of chimeric promoter activity using the gusA gene showed that it enhanced transgene expression levels in anthers of transgenic Brassica juncea (Indian oilseed mustard) plants by two- to three-fold over those obtained with either promoter alone but retained the temporal expression profile of the TA29 promoter. In the second strategy, the A9 and TA29 promoters were used to independently express two copies of the wild type barstar gene sequence within the same T-DNA borders. Alternatively, one of the promoters was used to express the wild type sequence and the other was used to drive expression of a codon-modified version of the barstar gene designed for enhanced and stable expression in dicot plants. The efficiencies of these constructs for fertility restoration was determined by retransforming a male sterile TA29-barnase line of B. juncea and analyzing the frequency and pollen viability of male fertile (restored) plants thus obtained. A significantly higher frequency of restored plants was obtained with constructs containing two transcription units of the barstar gene (92.9% for constructs with two copies of the wild type gene and 89.8% for constructs using the wild type and codon-modified sequences) as compared to that obtained with the chimeric promoter-barstar construct (77.8%) or the TA29-barstar construct (65.6%). Pollen viability assays on restored plants showed that the desirable extent of restoration is obtained in a higher proportion of male fertile plants generated using the construct containing a combination of the wild type and modified barstar genes. These observations were corroborated by genetic analysis of F1 progeny of crosses between various barstar lines and four different male sterile barnase lines, three of which could not be restored in earlier studies using TA29-barstar constructs. The simultaneous use of the wild type and modified barstar genes with the TA29 and A9 promoters was found to be the most effective method for fertility restoration with efficient restorers being identified for all the male sterile barnase lines tested, including those which could not be restored earlier. The two gene-two promoter strategy could be successfully deployed for enhancing and extending the tissue-specific as well as constitutive expression of other agronomically important transgenes.