Cloning Of SLs Signal Transduction Gene CsMAX2 In Cucumis Sativus L. And Verification Of Stress Resistance

Strigolactones?SLs?are carotenoid derivatives derived from the roots of plants.Since isolation of strigolactones from cotton roots,their synthesis,signal transduction,and physiological functions have been studied.Important progress.They have many important physiological functions,such as regulating the symbiosis of roots and arbuscular mycorrhiza,inhibiting plant branching,regulating plant flowering,and responding to abiotic stress.The MAX2 gene is a key gene in the strigolactone signal transduction pathway and encodes a class of F-box proteins.The MAX2 protein has shown the ability to regulate a variety of biological processes in plants,including plant structure,photomorphogenesis,aging,and karrikin Signal transmission,response to abiotic stress signals,etc.Cucumber is an important vegetable crop in China.Drought,salt and other abiotic stresses will seriously affect the growth,development and quality of cucumber.At present,there are few reports on the cloning of MAX2 gene in cucumber.Therefore,this experiment cloned MAX2 in cucumber.Gene,preliminary exploration of the molecular mechanism of this gene under drought and salt stress,provides a theoretical basis for the CsMAX2 gene to respond to abiotic stress in cucumber strigolactone signal transduction pathway.The test results show that:?1?Prediction and analysis of the structure and function of the protein encoded by the CsMAX2 gene through cloning and bioinformatics analysis.The results show that the protein translated by the open reading frame of this sequence has 715 amino acids.CsMAX2 is a class of hydrophilic proteins with a conserved F-box/LRR-repeat domain.It has the closest relationship to the MAX2 gene in pumpkin,and This protein interacts with proteins such as D27,CCD7 and CCD8 in the SL synthesis pathway.Promoter analysis revealed that the gene has multiple cis-acting elements that respond to abiotic stress and hormone responses.?2?Using real-time quantitative PCR technology to analyze the expression pattern of CsMAX2 gene in roots and leaves of cucumber under different tissues and organs and under different stress conditions.Analysis of expression characteristics showed that CsMAX2 was highly expressed in young stems and leaves of cucumber;CsMAX2positively induced up-regulated expression in cucumber roots and leaves in response to drought stress;under salt stress,CsMAX2 gene was up-regulated in cucumber roots.In response to salt stress,the expression in leaves was down-regulated;under ABA treatment,the CsMAX2 gene was up-regulated only when it was treated in leaves for 9 h,and the rest showed down-regulated expression.The results showed that CsMAX2 gene was involved in the response to drought,salt and ABA stress.?3?The CsMAX2 gene was overexpressed in Arabidopsis,and its response to salt,drought and ABA was verified.The CsMAX2 gene was constructed on the plant expression vector pCAMBIA2300,and the Agrobacterium tumefaciens-mediated inflorescence infiltration method was used to genetically transform Arabidopsis thaliana to obtain T₃ positive plants.The transgenic plants were further identified by qRT-PCR technology.Seeds of two CsMAX2 overexpressing Arabidopsis#2 and#3 strains were subjected to drought?250,300 mM mannitol?stress,salt?125,150 m M?stress,and ABA?0.3,0.4?M?treatment to compare the germination of each line The chlorophyll and malondialdehyde?MDA?contents of wild-type Arabidopsis and CsMAX2overexpressed Arabidopsis were also determined.The results showed that the germination rate of both the over-expressed Arabidopsis thaliana#2 and#3 lines was significantly higher than that of the wild type within 7 days after drought,salt,and ABA treatment,and the growth trend was more obvious.The chlorophyll content of CsMAX2overexpressed Arabidopsis is higher than that of wild type,and the content of malondialdehyde is lower than that of wild type.?4?To investigate the changes in the expression levels of marker genes and some genes in the abscisic acid signaling pathway under drought and salt stress.CsMAX2overexpresses 6 genes in Arabidopsis RD22,P5CS,COR15A,AREB1,DREB1A,and RD29A.The amount was significantly up-regulated,indicating that the CsMAX2 gene may regulate the expression of stress-related marker genes in response to stress responses.The abscisic acid pathway genes ABI1,ABI2,ABI4,ABI5,and NCED3 were significantly up-regulated under salt stress.Only ABI4 and ABI5 were inhibited,and ABI4,ABI5,and NCED3 were up-regulated under drought stress.This indicates that the stress tolerance induced by CsMAX2 may depend in part on the ABA signal transduction pathway.