The modified rice αAmy8 promoter confers high-level foreign gene expression in a novel hypoxia-inducible expression system in transgenic rice seedlings

Abstract

Expression of α-amylase genes in rice is induced not only by sugar starvation and gibberellin (GA) but also by O2 deficiency. Promoters of two rice α-amylase genes, αAmy3 and αAmy8, have been shown to direct high-level production of recombinant proteins in rice suspension cells and germinated seeds. In the present study, we modified the cis-acting DNA elements within the sugar/GA response complex (SRC/GARC) of αAmy8 promoter. We found that addition of a G box and duplicated TA box leads to high-level expression of αAmy8 SRC/GARC and significantly enhances αAmy8 promoter activity in transformed rice cells and germinated transgenic rice seeds. We also show that these modifications have drastically increased the activity of αAmy8 promoter in rice seedlings under hypoxia. Our results reveal that the G box and duplicated TA box may play important roles in stimulating promoter activity in response to hypoxia in rice. The modified αAmy8 promoter was used to produce the recombinant human epidermal growth factor (hEGF) in rice cells and hypoxic seedlings. We found that the bioactive recombinant hEGF are stably produced and yields are up to 1.8% of total soluble protein (TSP) in transformed rice cells. The expression level of synthetic hEGF containing preferred rice codon usage comprises up to 7.8% of TSP in hypoxic transgenic seedlings. Our studies reveal that the modified αAmy8 promoter can be applicable in establishing a novel expression system for the high-level production of foreign proteins in transgenic rice cells and seedlings under hypoxia.

Knockdown expression of a MYB-related transcription factor gene, OsMYBS2, enhances production of recombinant proteins in rice suspension cells

“…A strategy that combines all previous approaches is expected to further improve recombinant protein production in rice suspension cells. In addition to the αAmy3 promoter, a modified rice αAmy8 promoter has been used to produce a recombinant human epidermal growth factor in rice suspension cells and seedlings [ 45 ]. Knockdown of OsMYBS2 expression increases the mRNA level of αAmy8 in rice suspension cells [ 22 ], the promoter of which also contains a TA box [ 7 ].…”

Snorkeling Strategy: Tolerance to Flooding in Rice and Potential Application for Weed Management

“…Rice seeds that germinate well under low oxygen supply have higher expression of RAmy3D than in rice varieties intolerant to hypoxia [ 3 ]. Overexpression of RAmy3D gene in the coleoptile and endosperm of transgenic rice genotypes increased the germination capacity under anoxic conditions [ 16 ]. RAmy3D codes for alpha-amylase, which is involved in the catabolism of carbohydrates during germination.…”

“…An RNAi system composed of an unfitness gene together with another gene of interest, such as herbicide resistance, would reduce the spread of the transgene into weedy relatives. The gene silencing process has been pursued as an advanced biotechnological tool to reduce the gene flow consequences of transgenic crops [ 16 ], such as rice to weedy rice, sugar beet to wild beet, sorghum to johnsongrass, and canola to wild mustard [ 17 , 18 ]. In this case, the transgene silencer is inactive in the crop and is activated only in the weedy relative based on recognition of genetic differences in these plants.…”

Auxin Plays a Role in the Adaptation of Rice to Anaerobic Germination and Seedling Establishment

“…These two proteins interact and induce the MYBS1 transcription factor, subsequently activating the expression of starvation-induced α-amylase gene, αAmy3 / RAmy3D (Lee et al, 2009). The CPK15-SnRK1A-MYBS1-α-amylase signaling pathway has been demonstrated to be critical to sugar and oxygen deficiency in rice (Lee et al, 2009; Lee et al, 2014; Wu et al, 2014b). A trehalose-6-phosphate phosphatase gene, OsTPP7 , has been identified and demonstrated to promote the seedling establishment and enhance the AG tolerance in rice (Kretzschmar et al, 2015).…”