Zinc finger nucleases (ZFNs) have been used for targeted mutagenesis in eukaryotic cells. Custom-designed ZFNs can induce double-strand breaks (DSBs) at a specific locus. Our custom ZFN dimer was designed 3-finger of left and 4-finger of right with 2 kb size using 2A. A Ti-plasmid vector, pTA7002 containing the target site of SSS4A gene for a ZFN pair, that was shown to be active in yeast, was integrated in the rice genome. This promising technique for genome engineering was induced into 4 exon region of SSS4A gene in rice genome using Agrobacterium-mediated transformation. The SSS4A full-length cDNA was 5,070 bp consisting of a 318 bp 5′-untranslated region (UTR), a complete ORF of 2,928 bp encoding a polypeptide of 975 amino acids and a 3′-UTR of 1,824 bp. The vector is based on glucocorticoid receptor inducible gene expression system. Thus, SSS4A::ZFN expression was tightly controlled and the phenotype in low concentrations 10uM of the glucocorticoid hormone dexamethasone (DEX). In plant cells, transient ZFN expression is achieved by direct gene transfer into the target cells. For an alternative, ZFN delivery and production of mutant plants using a tobacco transient expression system for indirect transient delivery of ZFNs into a variety of tissues and cells of plants. ZFN activity was determined by PCR and sequence analysis of the target site. ZFN induced plants were obtained in up to 2% of the PCR products, consisting of deletions ranging between 1and 100 bp and insertions ranging between 1 and 10 bp. Our results describe an alternative to direct gene transfer for ZFN delivery and for the production of mutated rice.

UDP-glucose 4-epimerase (UGE; EC 5.1.3.2) is an enzyme that plays an essential role in the interconverts UDP-D-glucose (UDP-Glc) and UDP-Dgalactose (UDP-Gal). Five members of the Chinese cabbage (Brassica rapa) UDP-glucose 4-epimerase gene family, designated BrUGE1 to BrUGE5, have been cloned and characterized. Quantitative PCR shows that the BrUGE1and BrUGE4 mRNA are most abundant among other BrUGE genes, accounting for more than 55% of total BrUGE transcripts in most of the tissues examined. All genes showed organ specific expression pattern, two of which (BrUGE1 and 4) actively responded after Pectobacterium carotovorum subsp. carotovorum infection, while four genes (BrUGE-1, -3, -4 and -5)were shown to respond considerably against salt, drought and abscisic acid (ABA) treatments. To better understand the function of the UGE gene, we constructed a recombinant pART vector carrying the BrUGE1 gene under the control of the CaMV 35S promoter and nos terminator and transformed using Agrobacterium tumefaciens. We then investigated BrUGE1 overexpressing rice lines at the physiological and molecular levels under biotic and abiotic stress conditions. Bioassay of T3 progeny lines of the transgenic plants in Yoshida solution containing 120 mM Nacl for 2 weeks, confirmed that the BrUGE1 enhances salt tolerance to transgenic rice plants. Also T3 progeny lines of the transgenic plants, when exposed to infection caused by Xanthomonas oryzae pv oryzae, showed tolerance to bacterial blight. These results showed that BrUGE1 can be used as potential genetic resource for engineering Brassica with multiple stress resistance.

Wheat (Triticum aestivum L.) grain texture is an important determinant of milling properties and end product use. Two linked genes, puroindoline a (PINA) and puroindoline b (PINB), control most of the genetic variation in wheat grain texture. Wheat seed proteins were examined to identify PINA and PINB gene using two pre-harvest sprouting wheat cultivars; Jinpum (resistant) and Keumgang (susceptible).Wheat seed proteins were separated by two-dimensional electrophoresis with IEF gels over pH ranges: pH 3-10. A total of 73 spots were digested with trypsin resulting peptide fragmentation were analyzed by matrix assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF/MS). Mass spectra were automatically processed and searched through NCBInr, SWISS-PORT and MSDB database with mono isotopic masses and complete gene sequence were found by UniProt database. Puroindoline a and puroindoline b that is responsible for grain texture related with baking performance and roughness. Two spots were found Pin b (16.7 kDa) and Pin a (16.3 kDa) in Jinpum compare to seven spots were identified Pin a (16.1 kDa, 16.3 kDa) and Pin b (16.7 kDa, 9.5 kDa and 14.4 kDa) in Keumgang. Some selected spots were identified puroindoline like grain softness protein (16.9 kDa, 17 kDa and 18.1 kDa) in Keumgang. Moreover, to gain a better inferring the identification of puroindoline related proteins using proteomics, we accomplished a complete gene sequence of PINA and PINB gene in pre-harvesting sprouting wheat seeds between resistant (Jinpum) and susceptible (Keumgang).