Abstract:

【Objective】 High salt stress is one of the main factors affecting crop yield. Setaria italica L. is characterized by its strong tolerance to stress. Therefore, screening salt tolerant related genes from millet is important for salt stress tolerant improvement of crops through genetic transformation.【Method】 The expression profile of AP2/ERF like gene SiANT1 were analyzed in Yugu 1 at seedling stage under high salt, low nitrogen and PEG simulated drought conditions. Millet gene SiANT1 was transformed into rice Kitaake by agrobacterium-mediated transformation method. overexpressing SiANT1 rice seeds and wild type Kitaake were digested with 0.9% saline and tap water, respectively. The phenotypes of germinating stage and germination rate were observed. Rice seedlings were treated with 0.9% NaCl in Hogland nutrient solution for 10 days and then dried at 65℃ for 48 hours dry weights of all the rice overexpressing SiANT1 and wild type (WT) plants were analyzed. At the same time, phenotype of all plants was analyzed in the field and all plants were irrigated once with 0.9% saline before rice pregnancy. Under the similar salt treatment condition, single seed grain weigh plant height and tiller number per plant of the overexpressing SiANT1 rice lines and wild type were also analyzed. The real time-PCR method was used to verify the expression of SiANT1 and its possible downstream genes in transgenic rice lines.【Result】 Setaria italica L. SiANT1 protein (XP004985124.1, SiANT1) belongs to the AP2 subfamily and has a close genetic relationship with sorghum (XP021318293.1, SbANT1) and maize (XP008658933.1, ZmAP2).The gene of SiANT1 was induced by high salt stress (100 mmol·L^-1 NaCl) in Yugu 1. The SiANT1 was ligated to LP047 1118-Bar-ubi-EDLL vector, Agrobacterium transformation method was used to transfer SiANT1 into rice genome, positive T₀ plants were screened, then breeded two generations and got T₃ seeds. Under water immersion, the germination rate of SiANT1 overexpression rice was similar to that of wild type. Germination rate was more than 90%. Under 0.9% saline immersion, the overexpression of SiANT1 was significantly inhibited. Compared with the wild type, over-expression of SiANT1 rice seeds dew white was delayed, but finally(treatmented for 120h) germination rate was more than 80% .After treatment with 0.9% NaCl at seedling stage in greenhouse, the dry weight of single plant overexpressing SiANT1 increased 14.11%-37.42% more than that of WT, which had significant difference at the 1% level. The grain weight of transgenic rice overexpression SiANT1 in field increased 56.12%-76.58% higher than that of WT, which had significant difference at the 5% level, the number of tillers per plant and plant height increased, but there was no significant difference with the wild-type. Semi-quantitative RT-PCR analysis showed that all three rice lines overexpressing SiANT1 expressed SiANT1 at the RNA level. Real time-PCR results showed that the relative expression level of SiANT1 in three rice lines overexpressing SiANT1 was different but significantly increased compared with that of the recipient, and the relative expression levels of endogenous salt-tolerance-related genes OsSOS1 and OsZFP182 were 1.1-1.7 fold and 1.6-2.3 fold respectively higher than that of the recipient.【Conclusion】 It has been confirmed that OsSOS1 and OsZFP182 are salt tolerance related genes. SiANT1 gene has certain salt tolerance. Overexpression of SiANT1 in rice may increase salt tolerance by increasing the expression of downstream genes OsSOS1 and OsZFP182.

Key words: millet (Setaria italica L.), SiANT1 gene, AP2 / ERF transcription factor, salt tolerance, salt tolerant related genes