Selection And Verification Of Key Genes Involved In Salt Tolerance In The Suberin Lamella Of Grape Root

With the acceleration of the modernization process,soil salinization is becoming more and more serious all over the world.Hence,agricultural economy and environmental security are facing severe challenges.The salinized soil area is large and widely distributed in China.At present,planting salt tolerant crops is considered to be the most economical and sustainable way to improve saline alkali land.Grape has strong ability to various soil types and climatic conditions,and has strong tolerance to salt and alkali.It is of great significance to study the characteristics and mechanism of salt tolerance of grape,and to select excellent varieties of salt tolerant grape.The strong salt resistance of grape is related to the restriction of poisonous ions from root system.However,the mechanism of limiting toxic ions in grape roots is not clear.In this study,Crimson Seedless,and 1103P,a salt sensitive grape variety,were used as experimental materials to explore the mechanism of salt exclusion from physiological,cellular and molecular aspects,and to screen the key genes involved in salt exclusion.Through the bioinformatics analysis of the gene and the determination and comparison of the physiological indexes of the overexpressed plants and wild-type plants in germination and seedling stage under salt stress,the role of the gene in plant salt resistance was analyzed.The main experimental results are as follows:1. The participation of the suberin lamellae in salt exclusion was observedWith 0 and 50 m M Na Cl treatment on Crimson Seedless and 1103P,Section of roots(20,30,40 mm from root tip)were made and stained.The result of stained showed that the suberin lamellae of grape root was thickened in Na Cl treatment group,Which is particularly notable in Crimson Seedless,While,it was slightly weak in1103P.These results indicate that the ssuberin lamellae of grape root may be involved in its salt exclusion.2. The study of grape salt tolerance and the screening of related key genes by Fluorescence quantification.The RNA was extracted from Crimson Seedless and 1103P roots,stems and leaves treated with 50 m M Na Cl.In Crimson Seedless,the expression of thrombin related genes was low in the control group.The results showed that in Crimson Seedless,the expression of suberin related genes was low in the control group.After salt stress,kcs11-2,kcs11-3 and kcs4 all showed significant up-regulation expression,and the up-regulation expression of kcs11-2 showed obvious root tissue specificity.In the salt sensitive strain 1103p,the expression of suberin related genes was high in the control group,and showed root specificity.After salt stress,all related genes in roots were up-regulated slightly,but the degree of up-regulation was far less than Crimson Seedless.In conclusion,Vvkcs11-2 was selected as the gene of interest.3. The full length of Vvkcs11-2 was cloned in Crimson Seedless and 1103p respectively,and the sequence was used for bioinformatics analysis.The Vvkcs11-2 was cloned in Crimson Seedless and 1103p.The results of sequence alignment of two Vvkcs11-2 showed that there were seven different base sequences and one different amino acid sequence.According to the sequence of Vvkcs11-2,bioinformatics analysis was carried out by software such as Ex PASY,smart and tmhmm,which laid a theoretical foundation for further study on the function of Vvkcs11-2.4. Obtaining of over expressed Vvkcs11-2 Arabidopsis strains and analysis of its salt toleranceThe Vvkcs11-2 gene obtained from Crimson Seedless was cleaved by double enzymes and then positively connected to the p RI101.The p RI101-Vvkcs11-2 was transferred into GV-3101.The transgenic GV-3101 was used to infect Arabidopsis wild type,and then the over expressed strain was obtained.The overexpression lines(Vvkcs11-2-OE2?Vvkcs11-2-OE3?Vvkcs11-2-OE13)and wild types were treated with different concentrations Na Cl.Na Cl treatment inhibited the fresh weight,phenotype and main root length of Arabidopsis in varying degrees,but the inhibition effect of these treatments on wild-type Arabidopsis was more obvious,indicating that the overexpression of Arabidopsis at germination stage was more salt tolerant than that of wild-type Arabidopsis.After 0,100m M Na Cl treatment was used for different lines of Arabidopsis at seedling stage,physiological indexes such as ion content(mainly including Na~+and K~+,proline content and MDA content)in leaves were determined.The results showed that after salt treatment,the overexpression lines had higher K~+content in leaves,lower Na~+content and higher K~+/Na~+value;the content of MDA in overexpression group was lower and the difference of proline content was not significant,which indicated that the overexpression lines had strong salt tolerance ability in Seedling stage.These results showed that Vvkcs11-2could enhance the tolerance to salt stress by reducing the ion toxicity and increasing the osmotic stress substances such as proline.5. Screening of homozygous mutants of suberin lamellae related genes and analysis of their salt toleranceArabidopsis atkcs11(atkcs11)homozygous mutant was screened by"three primers".First of all,we screened the key gene Vvkcs11-2 of salt tolerance in grape,and atkcs11 was the homologous gene.Then,kcs11 homozygous mutant was treated with Na Cl,and it was found that the growth inhibition of kcs11 homozygous mutant was higher than that of Arabidopsis under salt stress.The phenotypic,fresh weight and main root length of Arabidopsis mutant and wild type were inhibited to varying degrees by the different concentrations Na Cl.Compared with wild-type strains,the mutant strains were more inhibited,which indicated that the salt tolerance of the mutant strains was relatively weak in the germination stage.The physiological indexes of ion content(Na~+and K~+,MDA content and proline content)in leaves of different Arabidopsis strains treated with 0and 100mm Na Cl at Seedling stage were measured.The results showed that under the salt treatment,the leaves of the mutant could maintain high K~+and low Na~+,and low K~+/Na~+;the MDA content of the mutant lines was higher than that of the wild type Arabidopsis,and the proline content was lower than that of the wild type Arabidopsis.These results show that the suberin lamella may play a role in salt tolerance of plants.