Arranged In Two Series In The Rice Gst Gene Function In A Preliminary Study

Based on the result of blast analysis, we isolated two glutathione-S-transferases (GSTs, E.C.2.5.1.18) genes, OsGSTZ1 and OsGSTZ2 from rice, which were two tandem-arranged GSTZ genes in chromosome 3 of rice. Semi-quantitative RT-PCR and northern blot analysis demonstrated that the expression of OsGSTZ2 up-regulated obviously in roots of rice and wasn't affected in leave after treatment with chlorsulfuron, the expression of OsGSTZ2 wasn't affected in roots and leave after treatment by abscisic acid (ABA), ethylene (ET) and methyl jasmonate (MeJA). The expression of OsGSTZ1 was constitutive in rice.The sequence analysis of genomic DNA for zeta class GST (GSTZ) locus in rice indicated that OsGSTZ1 and OsGSTZ2 were two high homologous GSTZ genes, the homology of which coding region nucleotide sequence and protein sequences were 76.87% and 68.85%, respectively. The upstream OsGSTZ1 included 8 introns and 9 exons, and encodes a protein of 243 amino acid residues, whereas OsGSTZ2 included 9 introns and 10 exons, and encodes a protein of 244 amino acid residues. The blastP analysis of animo acid sequence and analysis of protein function domain suggested they had certain characteristics of glutathione-S-transferases, and were two glutathione-S-transferases.We cloned the OsGSTZ1 and OsGSTZ2 from Oryza saliva ssp. Indica cv. "Guang Lu Ai No.4 by Invitrogen Gateway Technology system and express their proteins in yeast, respectively. We analyzed the activity of glutathione-S-transferases, the results showed the activity of glutathione-S-transferases in transgenic yeast was enhanced to 7-8 folds of the control yeast, which strongly suggested the OsGSTZ1 and OsGSTZ2 were glutathione-S-transferases.A 2,171 bp promoter sequence located upstream of the ATG translation start codon of the OsGSTZ2 gene was cloned, which hadn't typical TATA-box detected. Several truncated OsGSTZ2 promoters were generated by 5'-deletion, fused to the β -glucuronidase (GUS) coding sequence. The chimeric genes GSTZ2171::GUS, GSTZ1761::GUS, GSTZ962::GUS and GSTZ525::GUS were transformed into onion epidermal cells by Bio-Rad particle delivery system. In onion epidermal cells, the all promoter fragments are functional for promoting GUS expression. The GUS activity was detected in the seeds, leave and younger roots of the all transgenic rice plants; Later on, the GUS activity was barely detected in roots of GSTZ2171::GUS,GSTZ1761::GUS and GSTZ962::GUS rice plants. The results of GUS assay showed some enhancers located between positions -525 bp and -1 bp, -1761 bp and -962 bp and some repressors was located between positions -962 bp and -525 bp. GUS activity which was conferred by OsGSTZ2 promoters was lower and lower in roots and leave when the seedlings was older, the elements or motifs that lead in GUS activity attenuation along with the seedlings growing was located between positions -525 bp and -1bp. Expression of OsGSTZ2 is induced by various stimuli at the transcriptional level. OsGSTZ2 expression is induced by chlorsulfuron, glyphosate, SA and NAA, but isn't induced by ABA, ET and MeJA. Analysis of transgenic plants expressing the GUS gene under the control of different truncated OsGSTZ2 promoters revealed that cis-acting elements responding chlorsulfuron, SA and NAA were located between positions -962 bp and -525 bp, and that cis-acting elements responding glyphosate was located between positions -1761 bp and -962 bp. -1761 bp—-1 bp was a intact promoter.To investigate the function of GST, we generated transgenic rice plants expressing OsGSTZ1/2. Analyses of the transgenic rice seedlings by real-time reverse transcriptase-polymerase chain reaction (RT-PCR) and western blot showed the OsGSTZl/2 gene were over-expressed in transgenic rice. Their five T2 lines homozygous for the transgene were assayed, respectively, and had higher GST and glutathione peroxidase activities than non-transformants and control transformants. Transgenic rice contained lower levels of superoxide. Seedlings of the transgenic lines demonstrated greatly enhanced detoxify chlorsulfuron and glyphosate.Stress-response promoter is very useful to express special stress-tolerance gene in some sensitive plants. Semi-quantitative RT-PCR analysis demonstrated OsNRT1.1, a putative nitrate transporter gene in rice, is induced by drought. In order to check if the OsNRT1.1 promoter could response to drought stress, we cloned 2,019 bp upstream sequence of OsNRT1.1 gene, which had typical TATA-box and CAAT-box. Three truncated OsNRT1.1 promoter fragments fused to the β -glucuronidase (GUS) coding sequence were introduced into onion epidermal cells and rice plants, and all the constructs were functional for promoting GUS expression. NRT2019::GUS, NRT1196::GUS and NRT719::GUS showed same expression patterns in the transgenic rice, the blue color showed in all the seeds, roots, leaves and flowers. However, GUS activity conferred by different OsNRT1.1 promoters was upregulatedby drought up to about 3 times of the negative control, which indicated that OsNRT1.1 promoter is responded to drought stress, and the -719 bp to -1 bp of 5 - upstream sequence of the translation start codon (ATG) of OsNRT1.1 may be an intact promoter which included cis-acting elements required for drought response. The GUS activity didn't be affected by ABA, NaCl, (NH₄)₂SO₄, KNO₃ and Gln.