Identification Of ANK Family Genes In Foxtail Millet And Soybean And Function Analysis Of SiANK37 And GmANK114

The crop yield and quality are affected by various environmental stresses during their growth and development,especially drought and salt.In order to adapt to environmental change,a series of cascade reactions occur in plants,enhancing the expression of stress response genes to improve their stress tolerance.Therefore,it is very important to explore the functional genes and analyze the mechanism of plant stress tolerance.The Ankyrin（ANK）gene family is one of the largest known gene families in plants.Previous studies have found that ANK genes could respond to abiotic stress and positive regulate plant adaptation to environmental stress positively.As two important crops,the function of ANK family genes in foxtail millet and soybean is not completely clear.In this study,we identified the ANK genes in foxtail millet and soybean respectively.The biological functions and molecular mechanism of Si ANK37 and Gm ANK114 were investigated.The main research results are as follows:1.Identification of ANK family genes in foxtail millet and soybean genomes:A total of 124 and 226 nonredundant ANK genes were identified in the genome of foxtail millet and soybean respectively using the Pfam number（PF00023）of ANK domain.ANK family was classed into different subfamilies according to Conservative functional domain.There were seven ANK-IQ subfamily genes in foxtail millet genome,and proteins of these genes coding are localized in the nucleus.The q RT-PCR results showed that Si ANK37 was induced by drought,salt and ABA.In addition,17 ANK-RF genes were identified in soybean and multiple stress response elements were revealed in all Gm ANK-RFs promotors.Most of Gm ANK-RF genes were up-regulated by drought,salt and ABA,especially Gm ANK114.2.Analysis of function and regulatory mechanism of Si ANK37 in drought and salt tolerance:（1）Using Agrobacterium-mediated genetic transformation,Si ANK37 was overexpressed in rice and T3 overexpressed plants were obtained.Under drought and salt treatments,transgenic Si ANK37 rice had increased fresh weight of over-and under-ground,accumulation more proline,and accumulation less MDA,H₂O₂ and O₂^-,suggesting that Si ANK37 could improve the tolerance of transgenic rice to drought and salt.In addition,the expression of some stress-related genes was induced in transgenic rice,such as Os DHN1,Os RAB21,Os HSP82,Os ABI5,SAUR71,Os WRKY24,Os MYB4 and Os SNAC1.Further analysis showed that Si ANK37 protein had transcriptional activation activity and could enhance LUC activity mediated by Si MYB4 promoter.These results suggest that Si ANK37protein can positively regulate plant stress tolerance by regulating the expression of stress-related genes in plants.（2）Si ANK37 was used as a bait to screen the c DNA library of foxtail millet,and a serine-threonine protein kinase gene Si OSK1 was obtained.The physical interaction relationship between Si ANK37 and Si OSK1 was verified by yeast two-hybrid,pull-down and LUC complementation analysis.q RT-PCR showed that the expression of Si OSK1 was induced by drought and salt.Overexpression of Si OSK1 could improve the drought resistance of transgenic Arabidopsis.Therefore,it was inferred that Si ANK37 might play a role in improving drought tolerance of transgenic plants through interaction with Si OSK1.（3）The transgenic Si ANK37 wheat was obtained using Agrobacterium-mediated genetic transformation.Under drought and salt stress,the plant height of transgenic Si ANK37 wheat was higher than Fielder,and more proline and less MDA were accumulated in transgenic plant.The results showed that Si ANK37 could improve the tolerance of transgenic wheat to drought and salt.3.Function analysis of Gm ANK114 in drought and salt tolerance:The Gm ANK114 gene was transferred into soybean hairy roots by A.tumefaciens-mediated hairy root transformation technology to form the transgenic Gm ANK114 hairy root plants.Stress tolerance analysis showed that Gm ANK114 could enhanced drought and salt tolerance of transgenic soybean hairy roots.The content of proline in overexpressed hairy roots was higher,while the contents of MDA,H₂O₂ and O₂^-were significantly lower in overexpressed soybean hairy roots plants under drought and salt treatments than that in the control.In addition,the expression of stress-related genes were up-regulated in transgenic soybeans after drought and salt treatment,suggesting that Gm ANK114 improves drought and salt tolerance of plants by affecting the expression of stress-related genes.In this study,two stress-related genes Si ANK37 and Gm ANK114 were obtained by analyzing the ANK family genes of foxtail millet and soybean.Functional analysis showed that Si ANK37 and Gm ANK114 were involved in the response to abiotic stresses,and positively regulated the drought and salt tolerance of plants.The regulatory mechanism of plant stress tolerance of Si ANK37 and Gm ANK114 was preliminarily analyzed,which provided important clues for the study of drought and salt tolerance mechanism of ANK genes,and also provided a new research direction for the cultivation of stress-resistant crop varieties.