The Expression And Function Analysis Of The Salt Tolerance Gene SST In Rice

A salt tolerant mutant at seedling stage was obtained from radiation mutagenesis of an indica rice cultivar R401. The mutant phenotype was caused by a single recessive gene, designated sst. Gene mapping and sequencing analysis identify OsSPL10 as the candidate gene. GUS expression vector driven by OsSPL10 promoter and the positive complementary transgenic plants have been obtained. In this study, the identification of the phenotype of the positive plants was finished. qRT-PCR and GUS staining techniques have been used to analysis the expression level of SST. We also researched physiological response of the mutant in the germination and seedling stages under NaCl stress compared with the wide type R401. The physiological function of the gene was analyzed. The results as follows:(1) Genetic complementation experiments. The Ti generation of the complementary transgenic rice at 1.5-leaf stage was moved into a basin for liquid culture. The control groups were the wild type R401 and the mutant sst. After 10 days, they were given 100 mmol/L NaCl stress. The results showed that the mutant rices under stress for five weeks were survival, while the wild type were death. At this point, most of the Ti generation was death. The result showed that OsSPL10 is S5T gene.(2) The spatial and temporal expression analysis of SST with the qRT-PCR technology. The materials were the wild type R401 and the mutant sst. The RNA extracted from the seedlings ground part and root, flag leaf and its sheath, secondary leaf and its sheath, stem, more than 10 cm spikelet, and less than 5 cm spikelet of reproductive growth stage was used to finish the qRT-PCR analysis. The result showed that SST in the wild type has the highest expression level in less than 5 cm spikelet. It also has a high expression level in seedling shoot and root. The mutation sst expression level was down-regulated. These results may indicate that SST not only involved in the salt tolerance of rice but also be related to seedling nutrition growth and floral organ early development.(3) The spatial and temporal expression analysis of SST with the GUS staining technology. GUS vector has been previously constructed. We then transformed the recombinant vector into Nipponbare using the Agrobacterium.2 positive transgenic plants were obtained. Different tissues were taken for GUS staining in bud stage, seedling stage, booting stage, and heading stage. Results showed that SST expressed in the root, leaves, leaf sheaths, pulvinus, spikelet, epidermal hair, lemma, and flower organs of the four stages and the expression position was very specific. The result further described SST is a kind of pleiotropic gene which may be involved in the regulation of rice vegetative growth and reproductive development.(4) Salt stress effects on the expression of SST in the seedling stage. The materials were the wild type R401 and the mutant sst. The RNA of the seedlings ground part and root was extracted at the different time after 150 mmol/L NaCl stress. The time points include 0 h,0.5 h,1 h,2 h,4 h,12 h,24 h, and 48 h. The result showed that SST was finally induced in the wild type by salt stress. In the mutation, the gene of SST expression level was reduced and the mechanism of inducement of the gene was gone.(5) Physiological experiment at germination stage. The materials were the wild type R401 and the mutant sst. The control group was treated with water. The experimental group was treated with 150 mmol/L NaCl stress. Germination impetus was counted at the fourth day. Germination percentage and growth characteristics (shoot length, main root length, root number, fresh weight) were counted at the seventh day. The result showed that salt stress inhibited all the growth index of the wild-type and the mutant. There were no differences in the extent of inhibition of germination percentage, fresh plant weight, and shoot length in the wild-type and the mutant, but the Germination impetus, main root length, and root number were inhibited more seriously in the mutant than in the wild-type rice. The seedling stage salt tolerant mutant had no salt tolerance during the bud stage.(6) Physiological experiment at seedling stage. The materials were the wild type and the mutant. The control group was treated with nutrient solution. The experimental group was treated with 150 mmol/L NaCl stress. The physiological and ecological responses of the wild type and the mutant rice to salt stress were investigated at the different time, including fresh and dry weight, fresh weight water content, the contents of chlorophyll, carotenoid contents, the contents of PRO, MDA contents, and permeability of membrane. The NaCl stress induced less reduction of fresh and dry weight, fresh weight water content, and carotenoid contents in the mutant than that in the wild type, and the contents of free proline, MDA contents, and permeability of membrane of the wide type have a more increase under salt stress. There was no difference in the inhibition of chlorophyll content between the two. Under salt stress, the salt tolerance of the mutant may was not related to the molecular pathways, metabolic pathways or sphysiological pathways involved in the chlorophyll content. It is possible to achieve salt tolerance advantage by means of all possible pathways involving fresh weight, dry weight, water content, carotenoid content, MDA content, PRO content, and cell membrane permeability.