Functional study of a salt-inducible TaSR gene in Triticum aestivum.

The gene expression chip of a salt-tolerant wheat mutant under salt stress was used to clone a salt-induced gene with unknown functions. This gene was designated as TaSR (Triticum aestivum salt-response gene) and submitted to GenBank under accession number EF580107. Quantitative polymerase chain reaction (PCR) analysis showed that gene expression was induced by salt stress. Arabidopsis and rice (Oryza sativa) plants expressing TaSR presented higher salt tolerance than the controls, whereas AtSR mutant and RNA interference rice plants were more sensitive to salt. Under salt stress, TaSR reduced Na(+) concentration and improved cellular K(+) and Ca(2+) concentrations; this gene was also localized on the cell membrane. ß-Glucuronidase (GUS) staining and GUS fluorescence quantitative determination were conducted through fragmentation cloning of the TaSR promoter. Salt stress-responsive elements were detected at 588-1074 bp upstream of the start codon. GUS quantitative tests of the full-length promoter in different tissues indicated that promoter activity was highest in the leaf under salt stress. Bimolecular fluorescence complementation and yeast two-hybrid screening further showed the correlation of TaSR with TaPRK and TaKPP. In vitro phosphorylation of TaSR and TaPRK2697 showed that TaPRK2697 did not phosphorylate TaSR. This study revealed that the novel TaSR may be used to improve plant tolerance to salt stress.

Overexpression of the receptor-like protein kinase genes AtRPK1 and OsRPK1 reduces the salt tolerance of Arabidopsis thaliana.

AtRPK1 (AT1G69270) is a leucine-rich repeat receptor-like protein kinase (LRR-RLK) gene in Arabidopsis thaliana. The rice gene Os07g0602700 (OsRPK1) is the homolog of AtRPK1. AtRPK1 and OsRPK1 were overexpressed and the expression of AtRPK1 was inhibited by RNAi in A. thaliana. The functional results showed that the degrees of salt tolerance of the 35S:RPK1 A. thaliana plants were significantly lower than that of the control plants. The AtRPK1-RNAi A. thaliana plants exhibited higher salt tolerance than the wild-type plants (Col). The subcellular localisation results showed that the RPK1 proteins were mainly distributed on the cell membrane and that the overexpressed AtRPK1 proteins exhibited a significantly clustered distribution. The physiological analyses revealed that the overexpression of the RPK1 genes increased the membrane permeability in the transgenic A. thaliana plants. In response to salt stress, these plants exhibited an increased Na(+) flux into the cell, which caused greater damage to the cell. The real-time quantitative PCR analysis showed that the expression of the P5CS1 gene was inhibited and the SOS signalling pathway was blocked in the 35S:AtRPK1 A. thaliana plants. These effects at least partially contribute to the salt-sensitive phenotype of the 35S:RPK1 plants.

Function of the wheat TaSIP gene in enhancing drought and salt tolerance in transgenic Arabidopsis and rice.

Microarray analysis of a salt-tolerant wheat mutant identified a gene of unknown function that was induced by exposure to high levels of salt and subsequently denoted TaSIP (Triticum aestivum salt-induced protein). Quantitative PCR analysis revealed that TaSIP expression was induced not only by salt, but also by drought, abscisic acid (ABA), and other environmental stress factors. Transgenic rice plants that expressed an RNA interference construct specific for a rice gene homologous to TaSIP was more susceptible to salt stress than wild-type rice plants. Subcellular localization studies showed that the TaSIP localized to the cell membrane. Under conditions of salt and drought stress, transgenic Arabidopsis plants that overexpressed TaSIP showed superior physiological properties compared with control plants, including lower Na(+) content and upregulation of several stress resistance genes. Staining of transgenic tissues with ß-glucuronidase (GUS) failed to indicate tissue-specific activity of the full-length TaSIP promoter. Quantitative analysis of GUS fluorescence in transgenic plants treated with ABA or salt stress revealed that the region 1,176-1,410 bp from the start codon contained an ABA-responsive element and that the region 579-1,176 bp from the start codon upstream of the exon contained a salt-stress-responsive element. Based on these results, we conclude that the key part of the TaSIP gene is the region of its promoter involved in salt tolerance.

Overexpression of TaSTRG gene improves salt and drought tolerance in rice.

High salt and drought are the main factors affecting agricultural production. Thus, cloning stress-tolerance-related genes and identifying their functions are essential to enhancing crop tolerance to stresses. In this study, a salt-induced unknown wheat (Triticum aestivum L.) gene was identified and cloned according to microarray analysis of salt-tolerant wheat mutant RH8706-49 under salt stress. The gene was named Triticum aestivum salt tolerance-related gene (TaSTRG) and submitted to Genbank (Accession number: EF599631). TaSTRG expression in wheat is induced by multiple stresses including salt, polyethylene glycol (PEG), abscisic acid (ABA), and cold. Transgenic rice plants overexpressing TaSTRG gene showed higher salt and drought tolerance than the control. Under salt stress, the transgenic rice had a lower intracellular Na(+)/K(+) ratio than the control. Under salt and PEG treatments, these TaSTRG overexpressing rice plants had higher survival rate, fresh weight and chlorophyll content, accumulated higher proline and soluble sugar contents, and had significantly higher expression levels of putative proline synthetase and transporter genes than the control plants. These results indicate that the wheat TaSTRG gene could enhance plant tolerance to multiple types of stresses.

Cloning and functional analysis of wheat V-H+-ATPase subunit genes.

The root microsomal proteomes of salt-tolerant and salt-sensitive wheat lines under salt stress were analyzed by two-dimensional electrophoresis and mass spectrum. A wheat V-H(+)-ATPase E subunit protein was obtained whose expression was enhanced by salt stress. In silicon cloning identified the full-length cDNA sequences of nine subunits and partial cDNA sequences of two subunits of wheat V-H(+)-ATPase. The expression profiles of these V-H(+)-ATPase subunits in roots and leaves of both salt-tolerant and salt-sensitive wheat lines under salt and abscisic acid (ABA) stress were analyzed. The results indicate that the coordinated enhancement of the expression of V-H(+)-ATPase subunits under salt and ABA stress is an important factor determining improved salt tolerance in wheat. The expression of these subunits was tissue-specific. Overexpression of the E subunit by transgenic Arabidopsis thaliana was able to enhance seed germination, root growth and adult seedling growth under salt stress.

[Increasing the content of active constituents in Polygonum cuspidatum hairy root by gene transformation technology].

To increase the content of active constituent--RE and PD of Polygonum cuspidatum hairy root, through Ri-mediated gene transformation technology, modified high salt low pH method was used to distill genome DNA of grapevine (Vitis raparia). Primer was designed according to sequence of Genebank (AF128861). Through PCR amplification obtain RS gene sequence was obtained. Binary vector pCAMBIA1300-35S-RS was constructed. Frost thawing method was used to transform Agrobacterium rhizogenes ATCC11325. Scratched aseptic seedling leaf of Polygonum cuspidatum was contaminated subsequently. DNA conformity and mRNA expression of RS gene were investigated by PCR and RT-PCR respectively. RE and PD in transgenic hairy root were determined by HPLC. For the first time successfully inducement acquires transformed RS gene hairy root of Polygonum cuspidatum. Content of active constituents--RE and PD were 17 - 187 microg x g(-1) DW and 836 - 1 970 microg x g(-1) DW, respectively, the non-transgenic hairy root was 0 - 130 microg x g(-1) DW and 190 - 320 microg x g(-1) DW. In the different root selected, the content of PD was much higher than that in non-transformed hairy roots of Polygonum cuspidatum, the highest content is 5 times, but the content of RE has not increased apparently.

[Introduce Tagsk1 into salt-sensitive callus to improve the capacity of salt-tolerance by micropartical bombardment].

The Tagsk1 (Triticum asetium L. glycogen synthase kinase 1) gene derived from the genome of wheat salt-tolerance mutant RH8706-49 was cloned by PCR. The special primers designed according to full length cDNA sequence of Tagsk1 (AF525086). A binary expression vector pBI121-gsk1 containing Gus and Tagsk1 was constructed. And pBI121-gsk1 was introduced into the callus induced from mature embryos of salt-sensitive wheat H8706-34 and cv. China Spring by particle bombardment. The transformed callus were screened by Kanamycin and 0.5% NaCl. The salt-tolerance callus were obtained, which showed higher ability of salt-tolerance and could diffirentiate roots and buds on the medium containing 0.5% NaCl.

[Proteomic analysis of the salt tolerance mutant of wheat under salt stress].

Two dimensional electrophoresis was used to analyse the proteome of the salt-tolerant mutant of wheat (RH8706-49) and the salt-sensitive mutant of wheat (H8706-34) which had been treated by 1% NaCl for 72 hours. After being analysed by MALDI-TOF-MS and Mascot software, the qualitative and quantitative differences were identified between the two materials for five candidate proteins: H+-transporting two-sector ATPase, glutamine synthetase 2 precursor, putative 33 kD oxygen evolving protein of photosystem II and ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit. These five proteins are all belong to chloroplast proteins. They are likely to play a crucial role in keeping the function of the chloroplast and the whole cells when the plant was under salt-stress.

[Location of the fertility restorer gene for T-type CMS wheat by microsatellite marker].

Through the genetic analysis of a F2 population, derived from CMS line 75-3369A (T-type CMS wheat) and the restorer line 7269-10, the result indicated that the restorer line was conditioned by two dominant genes. A F2 population was used to map the fertility restorer (Rf) gene by microsatellite and BSA (bulked segregant analysis). Restorer and sterile DNA pools were established using the extreme fertile and sterile plants of F2 population, respectively. Among the 230 pairs of microsatellite primers, two markers were found polymorphic between the two pools. Linkage analysis showed that microsatellite marker Xgwm136 and Xgwm550 were linked with the two fertility restorer genes, respectively. One of the Rf gene was located on 1AS and the genetic distance between the SSR marker Xgwm136 and this Rf gene was 6.7 cM, the other Rf gene was located on 1BS and with a genetic distance of 5.1 cM to marker Xgwm550.

[Isolation and characterization of SIR73 gene fragment in salt resistant mutant of wheat involved in salt stress].

cDNA-AFLP (amplified fragment length polymorphism) is used to isolate genes differentially expressed in two wheat lines with the different resistance to NaCl derived from a single seed. A lot of cDNA fragments related to salt tolerance are obtained. Of with the number 73 cDNA fragment encodes for a transcription factors with an 32% similarity to human transcription factors in the relative amino acid which is named SIR73. Northern analysis confirms that SIR73 is strongly induced by NaCl stress and the expression in SR is more strongly induced than in SS.SIR73 may be involved in the regulation of gene expression in salt stress in wheat.