| LEA proteins (late embryogenesis abundant) are accumulated in abundance in seed,pollen and also induced by dehydration, osmotic stress, cold or exogenous abscisic acid.Usually, LEA proteins are supposed to be one of the proteins to involve in coping withunfavorable conditions for plants. According to the common amino acid sequence motifs and the conserved structuralfeatures, LEA proteins are classified into at least seven groups. Up to now, there are manyliteratures about the roles of group 3 LEA (LEA3) proteins in protecting stressed cells. Moreattention was paid on the research about the functional region and the salt tolerance of LEA3protein. It was reported that the expression of lea3 gene could confer salt and osmotic tolerancein transgenic rice, wheat and yeast recombinants as well. LEA3 proteins are characterized bya highly conserved 11-amino-acid repeating motif of "TAQAAKEKAGE". The motif waspredicted to form a putative amphiphilic α-helical structure, which could attenuate the damagecaused by high concentration of ions in dehydrating cells in such ways as binding water, or bysequestering or scavenging excess ions in cytoplasm of stressed cells. Soybean PM2 protein (Accession No.: M80664) is one of LEA3 protein families. Asreported by Hsing (1992), six copies of 22-mer amino acid repeating motif ofVNKMGEYKDYAAEKAKEGKDAT are present besides six copies of 11-mer amino acidmotif in PM2 protein sequence. After searching in Genbank database of NCBI, other sixembryonic or LEA3 proteins containing 22-mer repeating motifs were found out. The widelydistribution and highly conservatism of this 22-mer repeats implied its important role indevelopment of plant seeds. But to our knowledge, little information about the physiologicalroles of this 22-mer repeats in plant cells has been reported up till now. PM2 cDNA was isolated from soybean (Glycine max L. Merr.cv Bainong 6#) immatureseeds (35~45 days after flower). PM2 gene encodes a polypeptide consisted of 463aa. Thefull-length PM2 was cloned into pET28a vector, and then transformed into E. coli BL21 Starto get strain BL/PM2. The recombinants expressing truncated polypeptides of PM2A (aa1-262) or PM2B (aa 129-262, 22-mer repeating region), or artificial polypeptide PM2C(duplication of 22-mer repeating region) were also constructed according to the sequence ofPM2 protein. The results of SDS-PAGE and mass spectrometry approaches showed thatpolypeptide PM2,PM2A,PM2B and PM2C could expressed in E. coli, and the molecularweight was 54.46kDa,34.70 kDa,21.83kDa and 8.14kDa, respectively. These fusionpolypeptides were proved to be soluble in cytoplasm of E. coli, and to be heat-stable proteins. To validate the salt tolerance of expressed peptides, the transformants were cultured onthe plates with salt. Spot assays of BL/PM2 and BL/pET28 (as control) showed that proteinPM2 increased salt tolerance (500mM NaCl or 500mM KCl) of E. coli, rather than osmotictolerance (1100mM sorbitol). In addition, comparing the survival ratios of the transformantsunder 500mM NaCl or 500mM KCl stresses, the results showed that: 1) The survival ratiosof BL/PM2 and BL/PM2B were quite similar, both showing much higher than those ofBL/pET28. 2) The survival ratios of BL/PM2C were much higher than those of BL/PM2,BL/PM2A, and BL/PM2B. This provides the first experimental evidence that PM2polypeptide enhances salt tolerance of E. coli cells, and the 22-mer repeat region is animportant functional region. To further study the salt tolerance of polypeptides PM2,PM2A,PM2B and PM2Cfunctioned in high plant, the nucleotide sequences were cloned into pBI121 vector whichcould express aimed protein by the control of CaMV 35S promoter. The genes wereintroduced into tobacco by Agrobacterium tumefaciens. The genomic DNA was isolated fromtransgenic tobacco, and used for PCR as template. The results of PCR showed that the PM2,PM2A,PM2B and PM2C gene were integrated into the genomic DNA of transformed tobacco,and the frequency was 44.4%,75%,62.5% and 33.3%。 The transgenic and the control (with pB...
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