Functional Analysis Of Cysteine Rich Receptor-like Kinase ALS1 In Rice(Oryza Sativa L.)

Rice(Oryza sativa)is a major food crop in China,which is very important for food security in China.Rice will be subjected to various biological and abiotic stresses in the whole growth and development process.It is very important to enhance the resistance of rice to the stresses through breeding.Rice blast(Magnaporthe grisea)is one of the three major rice diseases.10%to 30%of the rice harvest is lost due to rice blast every year,which seriously threatens the food security.Soil salinization is one of the major abiotic stresses that restrict land utilization rate and lead to crop yield decline.It is widely existed in the world.Currently,about 20%of the world's land is salinized,and its salinized area increases year by year.ALS1 encodes a Cysteine rich repeat RLKs(CRKs),which is a of receptor-like kinases(RLKs).Most CRKs are differentially regulated by reactive oxygen species and play an important role in stress response,regulation of pathogen defense and programmed cell death(PCD).Functional analysis of ALS1 gene was conducted by protein sequence and evolutionary tree analysis,gene expression pattern analysis,subcellular localization analysis,ALS1-RNAi plant phenotype analysis,rice blast resistance analysis and salt stress resistance analysis.The results are as follows:1.ALS1 coding protein sequence and evolutionary tree analysisThe mutant als1 is a porphyroid dominant mutant.At the 4 leaf stage,the mutant als1 begins to show brown spots on the leaves and leaf sheaths and continues to mature.The older the leaf age and sheath age were,the more plaques were deposited.ALS1encodes a cysteine-rich receptor-like kinase CRKs containing an amino-terminal signaling peptide,two conserved CRR domains,and a conserved serine/threonine protein kinase domain.Phylogenetic tree analysis showed that ALS1 belonged to the highly conserved CRKs protein family.The results of subcellular localization indicated that ALS1 was located in the cell membrane,and the mutation of ALS1 did not change its localization.2.Analysis of the expression pattern of ALS1 geneThe rice leaves of seedling stage,tillering stage and heading stage were taken for qRT-PCR analysis.The results showed that ALS1 was mainly expressed in leaves and leaf sheaths,and the expression level in other parts was low.GUS protein staining experiments were performed on various tissues of Pro ALS1:GUS transgenic plants.The results showed that ALS1 was mainly expressed in the sieve tubes of vascular bundles in leaves and leaf shears.3.ALS1 is regulating rice resistance to rice blastThe wild-type,mutant als1 and ALS1-RNAi plants at seedling stage were inoculated with a physiological small species of rice blast.The results showed that compared with the wild-type,the mutant als1 plants had a smaller disease spot area and showed stronger disease resistance.The disease spot area of ALS1-RNAi plants was larger and the disease condition was more serious.The number of spores isolated from the leaves of wild-type and mutant als1 and ALS1-RNAi plants was highest in ALS1-RNAi plants and lowest in mutant als1.The expression of rice blast housekeeper gene MoPot2 was down-regulated in the mutant and up-regulated in ALS1-RNAi.Compared with the wild type,the resistance of mutant als1 to rice blast was enhanced and that of ALS1-RNAi to rice blast was decreased,indicating that ALS1 positively regulated the resistance of rice blast.4.ALS1 negative regulation resistance to salt stressThe wild-type,mutant als1 and ALS1-RNAi plants were treated with 200mM NaCl,200mM KCl and 20mM LiC solution for 7 days.The results showed that,compared with the control group,the wild-type plants died less and the leaves curled up,the mutant als1 died more and the leaves curled up more,and the ALS1-RNAi plants did not die and the leaves curled up less.Compared with the wild type,the mutant als1 treated by KCl showed more severe death,and the leaves of ALS1-RNAi plants curled up and died less.Wild-type and mutant als1 and ALS1-RNAi plants treated with LiCl were insensitive to LiCl.The results showed that ALS1 negatively regulated the resistance of rice to NaCl and KCl.The growth and development of wild-type and mutant als1 and ALS1-RNAi plants after stress treatment were statistically analyzed.The results showed that NaCl and KCl had significant inhibitory effects on plant height,while LiCl had no significant changes.The above results indicate that ALS1 is sensitive to Na~+and K~+.The ROS enzyme system and some resistance genes of wild-type and mutant als1and ALS1-RNAi after salt stress were quantitatively analyzed.The results showed that,compared with the wild-type,the expression levels of reactive oxygen scavenging enzymes in the mutant als1 were significantly down-regulated,and the expression levels in ALS1-RNAi were significantly up-regulated.The expression levels of reactive oxygen generating enzymes in the mutant als1 were significantly up-regulated,while the expression levels in ALS1-RNAi were down-regulated.It was speculated that the accumulation of reactive oxygen species in the mutant als1 was relatively high,while that in ALS1-RNAi was relatively low.Compared with the wild-type,the expression levels of some genes that play a positive regulatory role in salt stress resistance in mutant als1 were significantly down-regulated,and the expression levels in ALS1-RNAi plants were significantly up-regulated,indicating that the expression of some resistance genes in mutant als1 in response to salt stress was inhibited and activated in ALS1-RNAi.