| China is the third largest saline-alkali soil country in the world.Salinity is one of the most important abiotic stresses that affects the growth and development and causes yield reduction and extinction in maize.It is very important to excavate and identify genes related to salt tolerance and cultivate new salt-tolerant varieties to keep high and stable yield of maize,and to develop and utilize saline soil.In the early stage of the laboratory,a key candidate gene ZmCASPL2B2 was identified and cloned by using the maize leaf wrinkle mutant caspl2b2 material,and its allele mutant was created by gene editing technology.On this basis,ZmCASPL2B2 was overexpressed in rice and was found to significantly improve the salinity of rice.ZmCASPL2B2 knockout materials were used to confirm that it also regulated salt tolerance in maize.Furthermore,ZmPIP2;1 was identified as an interaction protein,and the possible mechanism of ZmCASPL2B2regulating the tolerance of maize was preliminarily analyzed.The main findings obtained were as follows:1.caspl2b2 mutant was sensitive to salt stress.The wild type（WT）and caspl2b2mutant material was treated with 200 m M NaCl in three-leaf stage,and found that after 3days of treatment,the leaves of caspl2b2 mutant wilted and shrunk,and the seedling height and shoot fresh weight were significantly lower than WT（P<0.05）.Salt stress inhibited photosynthesis in plants.Compared with the WT,caspl2b2 mutant had higher transpiration rate,stomatal conductance and intercellular CO2 concentration,and lower net photosynthetic rate（P<0.05）.After 7 days of salt treatment,the Na+content and Na+/K+ratio of caspl2b2 mutant were significantly higher than WT（P<0.05）,and the MDA content of caspl2b2 mutant was extremely higher than WT（P<0.01）.2.Evolution and expression pattern analysis of ZmCASPL2B2.Through the analysis of protein structure in website,ZmCASPL2B2 was a transmembrane protein with five transmembrane domains,and the first transmembrane domain had the highly conserved Arginine（Arg）of CASPL family.The genetic evolutionary analysis of the full length of ZmCASPL2B2 protein sequence showed that it had high homology with AtCASPL2B2.qRT-PCR result showed that ZmCASPL2B2 was expressed in response to salt stress,and with the prolongation of salt treatment time,the expression of this gene in leaves showed a trend of first decreasing and then rising,while the expression gradually decreased in roots.3.ZmCASPL2B2 could positively regulate the growth of rice and maize under salt stress.Overexpression of ZmCASPL2B2 significantly improved the salinity tolerance in transgenic rice,and the seedling height,shoot fresh weight and survival rate of overexpressed rice lines were significantly higher than the wild type under salt stress（P<0.05）,while there were no significant differences in the root traits of the underground part（P>0.05）.The MDA content of the wild type significantly increased（P<0.01）,which was 2-3 times than these of overexpression lines.The ZmCASPL2B2 gene knockout maize materials（KO1、KO2）were treated with 200 m M NaCl at the there-leaf stage for 7 d,and most of leaves were wrinkled and dried up,and the seedling height and shoot fresh weight were significantly lower than the non-transgenic lines（NT）（P<0.05）,while there were no significant difference between root length and total root weight（P>0.05）.The cross section of primary roots of NT,KO1 and KO2 materials were stained and observed,and found that there was lignified band thickening between endodermal cells at 3 cm-4 cm from the root tip,and the structure of Casparian strip in KO lines were intact,indicating that ZmCASPL2B2 did not affect the formation of Casparian strip.4.The accumulation of reactive oxygen species was increased in caspl2b2 mutant under salt stress.Transcriptome sequencing of leaves were treated with 200 m M NaCl for 4 h in WT and caspl2b2 mutant was performed,identified 849 differentially expressed genes,which were significantly enriched in glutathione metabolic process and oxidoractase activity.The RNA-seq data showed that compared with the wild type,there was no significant difference in the expression of ion transport-related genes（ZmHAK5,ZmHAK1,ZmHKT1,ZmHKT2 etc.）in the caspl2b2 mutant after salt treatment（P>0.05）,and the expression level of some genes were verified by qRT-PCR.There was also no significant change in the expression of genes（ZmESB1,ZmMYB36,ZmRBOHF etc.）related to the formation of Casparian strip（P>0.05）.Glutathione S-transferase family genes（ZmGSTs）were expressed at different degree of improvement.These results suggested that salt stress led to increased accumulation of reactive oxygen species in caspl2b2 mutant.5.The interaction of ZmCASPL2B2 and ZmPIP2;1 affected the salt tolerance in maize seedling stage.The interaction of ZmCASPL2B2 and ZmPIP2;1 was verified by yeast double hybridization experiment,and their interaction on the plasmic membrane was confirmed by bimolecular fluorescence complementarity experiments.qRT-PCR result showed that the expression of ZmPIP2;1 in knockout materials were significantly lower than that in non-transgenic lines（P<0.01）.From this,after interaction of ZmCASPL2B2 and ZmPIP2;1 in plasmic membrane,it participated in water transport process in plant,while the expression level of ZmPIP2;1 decreased in knockout materials,the ability of transport water decreased,and a high osmosis potential difference was formed,which affected the growth of maize under salt stress. |
