Enhanced resistance to two stem borers in an aromatic rice containing a synthetic cryIA(b) gene

Rice cultivars of isozyme group V include high-quality, aromatic rices that are difficult to improve by traditional methods because of the loss of quality characters upon sexual hybridization. Their low-tillering plant type predisposes them to economic loss from attack by stem borers, a group of insects to which they are susceptible. We report here the enhancement of stem borer resistance in cv. Tarom Molaii through transformation by microprojectile bombardment. Embryogenic calli derived from mature seeds were bombarded with gold particles coated with plasmid pCIB4421, carrying a synthetic truncated toxin gene based on the cryIA(b) gene from Bacillus thuringiensis, and plasmid pHygII, carrying the hygromycin phosphotransferase (hpt) selectable marker gene. Inclusion of 50 mg/l hygromycin B in culture media from bombardment through to rooting of plantlets eliminated escapes. The procedure generated three independent hpt transformants of which two also contained the cryIA(b) gene. One such line (No. 827) produced truncated (67 kDa) CryIA(b) protein equivalent to about 0.1% of total soluble protein. The cryIA(b) gene was controlled by the promoter of the maize C₄ PEP carboxylase gene and was expressed in leaf blades but was not expressed to a detectable level in dehulled mature grain. Line 827 contained about 3 copies of the cryIA(b) gene which segregated as a single dominant Mendelian locus in the second (T₁) and third (T₂) generations and co-segregated with enhanced resistance to first-instar larvae of striped stem borer (Chilo suppressalis) and yellow stem borer (Scirpophaga incertulas). T₂ line 827-6 homozygous for the cryIA(b) gene showed no dead hearts or whiteheads after infestation with stem borers, whereas T₂ line 827-25 lacking the gene averaged 7 dead hearts per plant and 2.25 whiteheads per plant. These results establish that transformation of high-quality rices of group V is a feasible alternative to sexual hybridization.     

The Histidyl-tRNA Synthetase from Streptococcus equisimilis: Overexpression in Escherichia coli,Purification, and Characterization

ABSTRACT

We describe the high-level expression of the Streptococcus equisimilis histidyl-tRNA synthetase gene hisS) in Escherichia coli and the purification and characterization of the gene product. Due to a lack of an efficient E. coli ribosome binding sequence in the hisS gene, the coding region was fused in-frame to the expression vector pT7-7, thereby creating a fusion gene construct (pT7-7recIII), which is under the control of a strong bacteriophage T7 promoter. Another construct (pT-7recII) was used for low level expression of the native histidyl-tRNA synthetase (HisRS). The plasmids were electroporated into E. coli HB101, which already contained pGP1-2. After temperature induction, the fusion HisRS, which has an extra 15 amino acids between the initiator Met and the second amino acid, Lys, was expressed at a level of —18% of total cell protein (～50 mg'liter of bacterial culture). The fusion HisRS was purified to >99% by a combination of anion exchange and cation exchange chromatography of the S100 fraction. The predicted MWs of the native and fusion proteins are 47,932 and 49,717, respectively. The mass of the active fusion HisRS was estimated to be 94,000 Da by Sephacryl S-200 gel filtration chromatography and 108,200 Da by nondenaturing PAGE. Both methods show that the funtional enzyme is a dimer of two identical subunits. SDS-PAGE analysis of purified fusion HisRS with or without reduction showed a single band of M_r = 53.7 kDa.