Screening Of Salt-tolerant Germplasm And Characterization Of Aquaporin Family In Radish

Radish(Raphanus sativus L.),as an important cruciferous root vegetable originated in China,has high nutritional value and is widely cultivated around the world.In the cultivation practice,the problem of soil salinization seriously affects the yield and quality of radish fleshy roots,which limits the development of the radish industry.It has becomed one of the important tasks to improve the salt tolerance of radishes and breed new salt-tolerance varieties.It is an important means to solve the problem of soil salt damage for carrying out evaluation of salt-tolerant germplasm resources,excavating radish salt-tolerance related genes,and revealing the molecular mechanism of radish salt-tolerance for solve the problem of soil salt damage.Plant salt stress related genes have also been identified one after another,and the research on the mechanism of salt stress response is relatively in-depth.As a type of integrated membrane protein that can efficiently transport water and other small molecule substrates,aquaporins(AQPs)can quickly respond to and promote the transport of water across the membrane when plants are subjected to salt stress,and maintain the water balance in the cell.Currently,AQP family has been identified in multiple species.However,The evaluation and screening system of salt-tolerant germplasm resources of radish has not been established yet,and the mining of genes related to salt tolerance of radish,especially whether the aquaporin gene can play a role in the response to salt stress is still unclear.In view of this,based on the establishment of a salt-tolerant germplasm evaluation system for radish seedlings,this study screened out salt-tolerant germplasm from 30 radish genotypes;based on the radish genome database,the AQP gene family was systematically identified,combined with transcriptome data and RT-qPCR analysis to mine AQP candidate genes related to salt stress response;and cloning and subcellular localization analysis of candidate genes,hoping to lay the foundation for genetic improvement of salt tolerance traits and salt stress response mechanism of radish.The main findings are as follows:1.Three kinds of radish genotypes ’XZH’,’XLM’ and’RG’ were treated with 6 different NaCl concentrations(0mM、100mM、150mM、200mM、250mM、300mM),and the relative plant height,relative root length,relative fresh weight above ground and relative fresh weight under ground were analyzed.The 250mM NaCl was determined as the appropriate salt stress concentration for radish seedlings.On this basis,30 seedlings of radish germplasm were subjected to salt stress treatment with 250mM NaCl,and their comprehensive salt tolerance evaluation values were counted.Using the principal component analysis method,the fresh weight under ground,root length and the root-to-root ratio can be used as the screening index for salt tolerance of radish seedlings.The salt tolerance of the germplasm was evaluated by systematic cluster analysis,and the results showed that TR3,TR12 and DF3 were highly salt-sensitive germplasm;TR16,TR17 and TR18 were highly salt-tolerant germplasm.2.Based on previous studies using transcriptomics and proteomics,it was found that the expression pattern of RsAQPs gene changed significantly after salt stress.It is speculated that aquaporin plays an important role in the regulation of salt tolerance of radish fleshy roots.In view of this,this study identified 61 RsAQPs genes at the level of the whole genome of radish,of which 57 RsAQPs genes were mapped to 9 chromosomes.Through comparative analysis of AQPs sequence of radish and Arabidopsis thaliana,RsAQPs genes were divided into four subfamilies,including PIP,TIP,NIP and SIP.Through transcriptome data analysis,it was found that the expression level of RsAQPs genes in radish at different development stages and different tissues were different.RT-qPCR analysis showed that the expression levels of RsPIP2-1,RsPIP2-5 and RsPIP2-8 in the roots and leaves of radish were significantly increased after 48 hours of salt stress treatment;while after 96 hours of treatment,RsPIP2-1,RsPIP2-5,RsPIP2-6 and RsPIP2-8 was significantly down-regulated in the root,RsPIP2-1,RsPIP2-5 were not significantly changed in the leaves,and RsPIP2-6 was significantly up-regulated in leaves.The above results indicate that some RsPIP2 genes may be the key gene for radish in response to salt stress.Additionaly,it was observed under a laser confocal microscope that both RsPIP2-1 and RsPIP2-6 proteins were located on the cell membrane by constructing a transient expression vector.