Study On Mechanisms Of Na~+/k~+Homeostasis In Sugar Beet(Beta Vulgaris L.)Different Ploidy Under Salt Stress

Soil salinization is becoming more and more serious due to human and nat ural reasons.Plants have developed various mechanisms to resist salt stress during long-term evolution.Carbohydrates,proline,antioxidant enzymes and some salt-tolerant genes play an important role in the salt tolerance of plants.Previous studies have been shown that different ploidy plants differ in salt tolerance.In order to ensure the normal growth of plants and increase crop yield,it is necessary to select plants that are more adaptable to the environment in crop breeding.In this study,the physiological characteristics and the expression patterns of functional genes(Bv NHX1,Bv SOS1,Bv HKT1;1,Bv AKT1,Bv HAK5 and Bv SKOR)related to Na~+and K~+uptake and transport were investigated in sugar beet(Beta vulgaris L.)cultivars―TY03410‖(tetraploid)and―TY03209‖(diploid)exposed to salt stress,and the Bv NHX1 gene editing vector was constructed by CRISPR/Cas9technology.The main results obstained from this study were as follows:(1)Under 300 mmol/L Na C l(high salinity),tetraploid accumulated less Na~+concentration,more K~+concentration,higher K~+/Na~+ratio,higher soluble sugar content,fructose content and sucrose content than diploid.That was under high salinity,tetraploid responsed to salt stress by maintaining the ion homeostasis and accumulating organic substance.(2)Under high salinity,malondialdehyde(MDA)content,proline content and peroxidase(POD)activity of tetraploid were significantly lower than diploid.Therefore,under high salt stress,maybe the extent of damage to the tetraploid was lower.(3)In tetraploid,under low salt(50 mmol/L)stress,Bv AKT1 and Bv HAK5 were significantly up-regulated by 3.9 and 6-fold at 24 h,respectively,in roots were down-regulated.Under high salt stress(200 and 300 mmol/L),Bv AKT1 was up-regulated 1.5-2.6 and 3.1-fold in leaves,respectively,and in roots were slightly up-regulated.In diploid,under different concentrations of salt stress,Bv AKT1 in leaves did not change significantly compared with the control,and Bv AKT1 in roots was down-regulated compared with the control.There was no significant difference in the expression level of Bv HAK5 between the roots and leaves of tetraploid compared with diploids.Thus,under high salt stress,Bv AKT1 mediated root K~+uptake in tetraploids and the ability of mediated K~+uptake was inhibited in diploid.(4)For Bv SKOR,compared with the control,under low salt stress,the expression level of root Bv SKOR in tetraploid and diploid were increased slightly and the difference was not significant;under high salt stress,the transcriptional abundance of the gene was increased significantly in tetraploid and diploid roots,but the expression level in tetraploids was obviously higher than in diploids.Indicated tetraploid Bv SKOR was more capable in transported K~+from roots to shoots.(5)For Bv HKT1;1,under low salt stress,the leaves and roots expression levels in tetraploid and diploid were increased significantly compared with the control,but there had no significant difference in two cultivars;however,under high salt stress,the expression abundance in tetraploid leaves and roots was significantly up-regulated and significantly higher than diploid.Indicated that under high salt concentrations,the tetraploid root Bv HKT1;1 reco verd excess Na~+through the phloem to protect the shoots from excessive Na~+.(6)The expression level of Bv SOS1 was mainly expressed in leaves and it was not significantly different at different salt concentration,the expression level of tetraploid Bv SOS1 was higher than diploid.Under low salt stress,leaves Bv NHX1 was up-regulated 2.1-fold in tetraploid at 24 h and 0.3-fold in diploid at 12 h;roots Bv NHX1 was increased 30%in tetraploid and decreased in diploid.Under high salt stress,tetraploid leaves and roots Bv NHX1 was significantly up-regulated compared with the control and the expression level was significantly higher than diploid.It was speculated that tetraploid roots and leaves was more capable of effluxing and regionalizing Na~+.(7)Selected 20 bases between the 3244-3263 bp in the fifth exon of gene Bv NHX1 as the target site,based on the intermediate vector p YLsg RNA-At U6-29 and the expression vector p YLCRISPR/Cas9-B,the Bv NHX1-sg RNA expression cassette was constructed by overlapping PC R and Method of Golden Gate Cloning,the ligation product of the p YLCRISPR/Cas9 vector and the Bv NHX1-sg RNA expression cassette digested with Bsa I-HF was obtained,respectively.