| Soil salinization is a major environmental factor limiting crop growth and yield.Saline-alkali land is an important reserve land resource in our country.However,it is largely barren due to its poor soil quality and low production efficiency,resulting in insufficient local agricultural production,which seriously restricts the development of regional economy.However,most crops are sweet soil plants that are sensitive to salt.Foxtail millet(Setaria italica L.),as a miscellaneous grain crop with Chinese characteristics,has become an important strategic reserve crop to cope with future water shortages due to its strong fibrous roots,well-developed root system,and strong salt and drought tolerance.Foxtail millet plays a very important role in ensuring food security in arid and barren areas.Screening and planting salt-tolerant crop varieties is one of the effective methods to reduce the damage of soil salinization.Currently,there are rarely report about the screening of key salt tolerance indicators and the salt tolerance evaluation of different foxtail millet germplasms from different regions,which seriously restricts the popularization and application of foxtail millet in saline-alkali land.In our previous research,942 foxtail millet germplasm resources were collected,and the agronomic traits,growth and development phenotypes in the field,and histochemical staining and some key physiological and biochemical indicators of these germplasms under normal and salt treatments conditions were detected and analyzed.The salt tolerance abilities of different varieties were evaluated based on the comprehensive characters and indicators.Further,the salt-tolerant and salt-sensitive varieties were selected for RNA-seq sequencing,and the key genes that may participated in salt tolerance were identified.The functions of one possible salt responsive gene SiRLK35 was further investigated by reverse genetics.The results are listed as follows:1.Based on the 942 core germplasms of foxtail millet,the field agronomic traits were analysed,and the preliminary salt tolerance screening of different varieties were carried out.Under salt treatment at seedling stage of hydroponic conditions,the foxtail millet varities with no significant phenotype variations were defined as salt tolerance varities,while the phenotypes such as leaves turned into yellow and wilt were defined as salt-sensitive compared with those of control groups.Thus,92 foxtail millet varities belong to salt-tolerant and salt-sensitive characters separately were obtained.We further performed peroxidative staining histochemical analysis,salt stress-related malondialdehyde content,electrical conductivity,POD activity,GST activity and other physiological indicators using the different salt-tolerant foxtail millet varieties obtained from the aboved screening processe,and finallyobtained about 10 salt-tolerant germplasms including Qiuyumao etc,and 11 salt-sensitive germplasms including Baigu and other varieties.2.The different expressed genes(DEGs)between salt-tolerant and salt-sensitive varieties were compared using RNA-Seq.29,291 DEGs were identified,of which28,547 were functionally annotated while 744 were not.Among these annotated DEGs,1067 were unique salt stress-responsive genes in salt-tolerant cultivars,and 1768 were specific salt stress-responsive genes in salt-sensitive cultivars.The GO and KEGG enrichment analysis showed that DEGs,mainly involved in organic acid metabolism,keto acid metabolism,carboxylic acid metabolism,small molecule metabolism,redox process and signal transduction under the 250 mmol·L-1Na Cl treatment,played key role in the response to salt stress in foxtail millet seedlings.The DEGs of salt tolerance and sensitivity are mainly enriched in metabolic processes,secondary metabolite synthesis and other metabolic pathways.3.Using CRISPR/Cas9 gene editing technology,two targeted sites in the SiRLK35gene of foxtail millet cultivar Ci846 were designed.The p C1300-UBI:Cas9-SiRLK35gene editing vector and SiRLK35 gene overexpression vector were constructed,and were transformed into the callus of foxtail millet by agrobacterium mediated system.The SiRLK35 overexpression and gene editing mutant of foxtail millet were obtained and then were used for phenotype analysis and some stress-related physiological indicators detecting under salinity.The results showed that the growth of SiRLK35transgenic and the control foxtail millet seedlings were all inhibited under salt stress,but the growth inhibition degree of SiRLK35 overexpression lines was lower than that of the control,and the inhibition degree of gene editing mutant plants were significantly higher than that of control.And both SOD and POD activities showed that the overexpression lines had higher scavenging ability of reactive oxygen species under salt.Summary of all,the salt tolerance of the SiRLK35 overexpression lines in foxtail millet was significantly improved,while the salinity tolerance of the SiRLK35 gene CRISPR/Cas9 knockout lines were significantly decreased at the germination and seedling stage,indicating SiRLK35 indeedly involved in salt responsive processe.This study provided excellent germplasm resources for the development and utilization of saline-alkali land,and provided candidate gene resources for salt-tolerant crop breeding.The desection of the SiRLK35 regulationin mechanisms laied the foundation for further molecular design breeding,and also laied a foundation for breeding new salt-tolerant foxtail millet varieties with excellent comprehensive characters. |
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