Abstract
Drought is one of the major abiotic stresses threatening rice (Oryza sativa) production worldwide. Drought resistance is controlled by multiple genes, and therefore, a multi-gene genetic engineering strategy is theoretically useful for improving drought resistance. However, the experimental evidence for such a strategy is still lacking. In this study, a few drought-responsive genes from rice were assembled by a multiple-round site-specific assembly system, and the constructs were introduced into the rice cultivar KY131 via Agrobacterium-mediated transformation. The transgenic lines of the multi-gene and corresponding single-gene constructs were pre-evaluated for drought resistance. We found that the co-overexpression of two genes, encoding a constitutively active form of a bZIP transcription factor (OsbZIP46CA1) and a protein kinase (SAPK6) involved in the abscisic acid signaling pathway, showed significantly enhanced drought resistance compared with the single-gene transgenic lines and the negative transgenic plants. Single-copy lines of this bi-gene combination (named XL22) and the corresponding single-gene lines were further evaluated for drought resistance in the field using agronomical traits. The results showed that XL22 exhibited greater yield, biomass, spikelet number, and grain number under moderate drought stress conditions. The seedling survival rate of XL22 and the single-gene overexpressors after drought stress treatment also supported the drought resistance results. Furthermore, expression profiling by RNA-Seq revealed that many genes involved in the stress response were specifically up-regulated in the drought-treated XL22 lines and some of the stress-related genes activated in CA1-OE and SAPK6-OE were distinct, which could partially explain the different performances of these lines with respect to drought resistance. In addition, the XL22 seedlings showed improved tolerance to heat and cold stresses. Our results demonstrate that the multi-gene assembly in an appropriate combination may be a promising approach in the genetic improvement of drought resistance.
Highlights
Drought, one of the major abiotic stresses which was faced by the ancestors of modern rice cultivars about ten thousand years ago, is still the major limiting factor of modern rice (Oryza sativa) production worldwide, and will still be in the foreseeable future (Sweeney and McCouch, 2007; Lobell and Tebaldi, 2014; Lesk et al, 2016)
Our studies revealed that the abscisic acid (ABA)-responsive bZIP transcription factors OsbZIP23 and OsbZIP46 play essential roles in drought resistance in rice, and these transcription factors can be activated by SnRKs including SAPK2 and SAPK6 (Xiang et al, 2008; Tang et al, 2012; Zong et al, 2016)
Drought resistance was pre-evaluated in the T1 generation in a paddy field facilitated with a movable rain-off shelter to find candidate multi-gene overexpression lines with improved drought resistance compared with the corresponding single gene overexpression lines and negative transgenic plants (KY131-N) for further study
Summary
One of the major abiotic stresses which was faced by the ancestors of modern rice cultivars about ten thousand years ago, is still the major limiting factor of modern rice (Oryza sativa) production worldwide, and will still be in the foreseeable future (Sweeney and McCouch, 2007; Lobell and Tebaldi, 2014; Lesk et al, 2016). Since rice is responsible for the feeding of a large part of the world’s population, great efforts have been directed to the genetic and molecular studies and the improvement of its drought resistance in recent decades (Luo, 2010; Hu and Xiong, 2014). The generation of a rice cultivar with enhanced abiotic stress resistance is achieved by two approaches: classical genetics and reverse genetics. The former approach, which consists of trait identification, mapping, and elite line construction, is known for being labor intensive and timeconsuming (Luo, 2010; Khan et al, 2016). With the advent of the genomics era, studies on the molecular mechanisms of abiotic stress responses in rice have been substantial, and a large number of candidate genes have been characterized, especially in recent years (Hu and Xiong, 2014; Yoshida et al, 2014; Todaka et al, 2015; Khan et al, 2016)
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