Functional And Regulation Pathway Analysis Of Two Salt-Stress Related Genes SiNF-YA5 And SiATG4 In Foxtail Millet

Salt stress is one of major abiotic factors to damage the growth and yield of crops. Foxtail millet origins from China, and has typical characteristics of drought and barren resistance in monocotyledon plants. Thus, all of these suggest that it may be act as an ideal material to study stress-resistant mechanism in monocotyledon. On the other hand, although it was enrichment in the source of foxtail millet in the native locus of china, the research of stress-resistant functional genomics is just started. Thus, it has important theoretical and practical significance for molecular breeding in crops through screening ideal resistance genes from the varieties of foxtail millet in china. As we known that autophagy widely existed in eukaryotic organisms, and the denatured or damaged organelles and and large molecules in the cell were degraded and subsequently reused to maintain the stability of metabolism. Also these biochemical processes played an important role in responding to extracellular stress and resistance environmental changes. However, the functional analysis of NF-Y like transcription factors and autophagy-related genes in regulating salt stress response is limited reported in plants. In our study, we have screened two candidate genes of foxtail millet, including NF-Y like transcription factor gene SiNF-YA5 and autophagy related gene SiATG4 through analysis of the transcriptome sequencing results, involved in responding salt stress. In addition, we systematically analyses their basic characteristics, expression profiles, biological functions and regulation pathway. The main results are as follows:1. Functional and regulation pathway analysis of an NF-Y like transcription factor gene SiNF-YA5.An NF-Y like transcription factor gene SiNF-YA5 was isolated from foxtail millet variety longgu 25. The full-length of SiNF-YA5 is 924 base pair(bp), encoding 307 amino acids, and the molecular weight of SiNF-YA5 protein is 33.6 kilo Dalton(kD). SiNF-YA5 was localized on the plasma membrane and nucleus in plant cell. Expression profiles analysis showed that SiNF-YA5 was induced by high salt, drought and low nitrogen stress, but not by ABA treatment. We constructed SiNF-YA5 transformation vector and over-expressed SiNF-YA5 in Arabidopsis, and then analyzed the salt tolerance of transgenic plants and wild-type during seed germination and seedling stages. According to the results, the rates of seed germination and green seedling of transgenic Arabidopsis were significantly higher than those in the wild-type Arabidopsis(WT) under high salt treatment(P < 0.01). During the stage of seedling development, the root development of transgenic plants overexpressing SiNF-YA5 in Arabidopsis is healthier compared to WT. The root surface area(P < 0.01) and plant fresh weight(P < 0.05) of transgenic Arabidopsis were significant higher than that of WT, respectively. These results indicated that SiNF-YA5 can confer salt tolerance of transgenic plants. Gene expression analysis showed that expression level of salt-stress related genes NHX1 and LEA7 in transgenic Arabidopsis increased significantly under high salt stress treatment compared to WT. It was previously reported that NHX1 is a kind of Na+/H+ transporters to transfer intracellular sodium ions to vacuole, while LEA7 is a kind of late embryonic development protein. These results indicated that SiNF-YA5 can enhance the salt tolerance of transgenic Arabidopsis by ion compartmentation or protecting protein activation. On the other hand, under ABA treatment, the transgenic Arabidopsis showed the similar sensitivity to ABA compared with WT, and there are also no obvious phenotypic changes during germination stage and seedling stage. Collectively, all of these results showed that SiNF-YA5 can enhance salt tolerance through ABA-independent pathway in transgenic plants.2. The characteristic and functional analysis of an autophagy related gene SiATG4.An autophagy related gene SiATG4 was isolated from millet variety longgu 25. The full-length of SiATG4 is 1443 bp, encoding 480 amino acids, and molecular weight of SiATG4 protein is 52.29 kD. SiATG4 was localized on the plasma membrane and nucleus in plant cell. Expression pattern analysis showed that SiATG4 was induced by high salt and drought stresses, as well as ABA. We constructed SiATG4 overexpression rice and then identified its salt resistance during seed germination and seedling stages. According to the results, the germination rate of transgenic rice was significantly higher than that of wild rice(CK) under high salt stress. During seedling stage, the results showed that transgenic rice were healthier than CK, and plant fresh weight was significantly higher than that of CK(P<0.01). These results showed that SiATG4 can enhance the resistance to salt and drought resistance in transgenic plant.In short, through this study we can not only provide new excellent candidate gene for crop resistance molecular breeding, but also offer an insight into the regulatory module and basement in stress response in foxtail millet.