Clones And Functional Analysis Of Vesicle-Associated Gene In Limonium Bicolor

Salt stress is a major abiotic stress in the world,which affects plant growth,reduces agricultural productivity and restricts regional development.However,as an important reserve land resource of our country,the value of saline land is unignorable.The exploitation and utilization of saline land is particularly important to solve the shortage of land resources in China.The most economical and effective measure for the exploitation and utilization of saline land is to use the salt-tolerant plants that can survive in saline land.Therefore,it is important to study the salt-tolerance mechanism of halophytes.Although there have been many successful experiences with halophytes at home and abroad,the salt-tolerance mechanism of halophytes still needs further study.As a typical recretohalophytes,the salt secretion mechanism is still unclear.Therefore,this paper cloned the genes that may be related to the secretion of salt of Limonium bicolor and analyzed their functions.In this study,three vesicle-related genes were cloned from the leaves of Limonium bicolor and the functions of these three genes were further studied:?1?Bioinformatics analysis of the three genes.?2?Analysis of overexpression in Limonium bicolor.?3?The genes in Limonium bicolor were knocked out by CRISPR-Cas9 technology and then the function of transgenic plants will be tested.?4?The analysis of overexpression in Arabidopsis.?5?To study mutant plants lacking vesicle transport related genes in Arabidopsis thaliana.The main results are as follows:1.Based on the results of RNA-Seq,we have obtained three vesicle-associated genes,which were LB00297,LB11044 and LB11277,respectively.According to the intermediate sequence of genes,the full length of the genes was obtained by 3'and 5'RACE techniques.The fragment size of the LB00297 gene is 1066 bp,the coding sequence of the gene is 660 bp,encoding 219 amino acids.The size of LB11044 gene is 1304 bp,the coding sequence of the gene is 708 bp,encoding 235 amino acids.The size of the LB11277 gene is 1478 bp,the coding sequence of the gene is 717 bp,encoding 238 amino acids.The three genes of LB00297,LB11044 and LB11277 were compared with the vesicle-associated genes in Arabidopsis thaliana,and the homology was 78.94%,59.92%and 67.92%,respectively.The physicochemical properties of proteins are analyzed.The proteins of LB00297,LB11044 and LB11277are all hydrophilic proteins,in which LB00297 is a stable protein,and LB11044 and LB11277 are unstable proteins.SMART analysis of the protein sequence found that LB00297 protein,there is a region of Longin and transmembrane rgion,and Longin region is regulation of SNARE-like regions.It is consistent with the structure of Arabidopsis vesicle transport proteins.There are coiled coil domains and transmembrane helical regions in LB11044 protein.LB11277 proteins:there are low complexity regions and transmembrane helix regions.Using the MEGA software to compare the homologous sequences of the genes,it was found that they were closely related to Spinacia oleracea and Arabidopsis thaliana.2.The CDS regions of the three genes LB00297,LB11044 and LB11277 in Limonium bicolor were connected to the expression vector pCAMBIA1300respectively,and the overexpressed plants were obtained by infecting leaves.The resistance of the expression vector was hygromycin.Therefore,it is a screening marker.The plants were cultured until they were homozygous and the phenotype under salt stress was observed.At the same time,the CRISPR-Cas9 technology was used to target gene modifications and the CRISPR-Cas9 vector was constructed.Using the method of infecting the leaves of Limonium bicolor,the three genes,LB00297,LB11044,and LB11277 were knocked out.In addition,the plants were cultured.3.The CDS regions of the three genes LB00297,LB11044 and LB11277 in Limonium bicolor were connected to the expression vector pCAMBIA1300respectively.The overexpression of Arabidopsis thaliana plants was obtained by inflorescence infestation and screened by hygromycin resistance.The transgenic seedlings were then tested at the molecular level.Finally,homozygous plants were identified according to Mendel's genetic separation law.The pure and overexpressed plants of Arabidopsis thaliana with vesicle-associated genes was obtained.The homozygous plants were named 97S-1,97S-6,44S-2,44S-5,77S-6 and 77S-7.SALK₀68951.56.00._X,SALK₀68950.54.70._X,SALK₀82847.19.80.n,SALK₁47591.51.65._X,SAIL₁21_G12.v1 and SALK₁19355C were selected as materials.The pure plant of Arabidopsis thaliana was obtained and detected at DNA level by PCR.4.Effect of salt stress on overexpression of Arabidopsis,wild type Arabidopsis and mutant homozygosity:the seeds of Arabidopsis,wild type Arabidopsis,and mutant homozygous plants were put on the medium with different salt content,and the germination of the seeds was observed.The results showed that under salt stress,the germination and growth of seeding were affected by different degrees of salt stress,and with the increase of salt concentration,the germination and growth showed a downward trend.However,the germination potential and germination rate of 97S-1,97S-6,44S-2,44S-5,77S-6 and 77S-7 were higher than wild type plants and mutant plants.The germination index showed that the overexpressing plants germinated faster and more neatly.The phenotypes of 77S-6 and 77S-7 were more obvious.Under salt stress,the root length of the over-expressed plantlets was significantly higher than that of the mutant plants,and there was no significant difference compared to the wild type plants.The salt tolerance of the overexpressed plants was slightly higher than that of the wild type,while the salt-tolerance of Arabidopsis lacking vesicle transport related genes was inferior to that of the wild-type plants.