| Soil salinization is a worldwide problem,threatening the growth and yield of crops,and hindering the sustainable development of modern agriculture.Exploring the salt-tolerant mechanism of halophytes,digging out key genes for salt-tolerance,and using modern biotechnology to cultivate salt-tolerant crops is the fundamental way to utilization of saline soils.Limonium bicolor L.can secrete excess of salt out of plant body through salt glands.If the key genes controlling salt gland development can be determined and heterologously expressed in non-halophytic plants such as crops,the salt tolerance of the transgenic plants,especially crops,will be improved.Earlier researches on salt glands mainly focus on tructures and salt secretion,but few studies have been done on genes that control the development of salt glands.The sequencing of the whole genome and RNA-Seqs of Limonium bicolor(Bag.)Kuntze have been done,and the candidate genes controlling salt gland development and salt secretion have been screened in our laboratory.Very interestingly,a large number of unknown functional genes with specific and high expression in the early stage of leaf development has been found.According to the maturation of the salt glands in stage B of the leaf,we guess the genes that control the development of salt glands are highly expressed in the stage A.In the current paper,three unknown functional genes that are highly expressed in the A and B stages(Lb2G14763,Lb3G18904 and Lb7G32827)were used to examine their functions in salt gland development and salt tolerance of Arabidopsis.The main results are as follows:(1)The Lb2G14763 gene is 690 bp and encodes 229 amino acids.It is found that the function of Lb2G14763 is unknown via blasting in NCBI.Bioinformatics analysis shows that Lb2G14763 protein has a possible transmembrane region(206-228).The results of subcellular localization experiments show that the protein encoded by the Lb2G14763 gene localizes to the plasma membrane.The results of phylogenetic tree analysis showed that the Lb2G14763 gene was closely related to an unknown gene of Moraxella and Alkanephagus,but was farther related to genes such as Osmanthus.The promoter sequence of Lb2G14763 was successfully cloned.Sequence analysis showed that the promoter of Lb2G14763 gene contains not only core promoter element(TATA-box),common cis-acting element(CAAT-box),but also hormone-regulated elements such as ABA cis-acting element(ABRE),jasmonic acid response element(TGACG-motif),gibberellin response element(GARE-motif),and light response element(GATA-motif).According to the analysis of promoter elements,two abiotic stresses(drought stress,salt stress)and three hormones(ABA,JA,GA)were used to treat seedlings of Limonium bicolor.The results showed that Lb2G14763 gene is involved in salt stress and drought stress response.Moreover,it is induced by IAA,JA and GA,indicating that the Lb2G14763 gene may play a certain role in the salt tolerance and drought resistance of plants through the signal transduction pathway mediated by IAA,JA and GA.In order to further understand the function of the Lb2G14763 gene,the recombinant expression vector p CAMBIA3301-35S-GUS-Lb2G14763was constructed,and the p HEC401-CRISPR-Cas9 knockout vector was constructed at the same time.The construction of this part of the vector laid the foundation for further study on its function in salt gland development and stress tolerance of Limonium bicolor in the future.Heterologous transformation of Lb2G14763 in wild-type Arabidopsis thaliana was performed and transgenic T3 homozygous seeds were used to determine its function in salt tolerance.Compared with wild-type Arabidopsis under the same concentration of salt treatment,germination rate,cotyledon growth rate and the root length of Lb2G14763-overexpressed lines were all lower;More Na~+but less K~+,and the higher ratio of Na~+/K~+was found in Lb2G14763-overexpressed lines;the expression of salt tolerance related genes such as SOS1,NHX1,At GSTU5,At P5CS1 is lower in Lb2G14763-overexpressed lines than that in wild type,indicating that Lb2G14763 negatively regulates plant salt tolerance.Interestingly,when the Lb2G14763 gene was heterologously expressed in Arabidopsis,the number of epidermal hairs in the transgenic lines decreased in a dose dependent mode,indicating that this gene is related to epidermal hair development.(2)The Lb3G18904 gene is 501bp and encodes 166 amino acids.It was found that the function of Lb3G18904 is unknown via blasting in NCBI.The results of bioinformatics analysis showed that the Lb3G18904 protein has a transmembrane domain(69-91)and a coiled-coil domain(115-149).The results of subcellular localization experiments show that the protein encoded by the Lb3G18904 gene localizes to the cell membrane.The phylogenetic tree analysis showed that the Lb3G18904 gene is closely related to an unknown gene in elm,walnut,and pumpkin,and has a farther relationship with genes such as quinoa,sugar beet,and spinach.The promoter sequence of Lb3G18904 gene was successfully cloned.Sequence analysis showed that the promoter of Lb3G18904 gene contains not only core promoter elements(TATA-box),common cis-acting elements(CAAT-box),but also hormone-regulated elements such as ABA cis-acting element(ABRE),jasmonic acid response element(TGACG-motif)and light-responsive element(GATA-motif).According to the analysis of promoter elements,2 abiotic stresses(drought stress,salt stress)and 4 hormones(ABA,JA,GA,IAA)were used to treat seedlings of Limonium bicolor.The results showed that Lb3G18904 gene is involved in salt stress and drought stress response.Moreover,it is induced by IAA,JA,GA and IAA,indicating that the Lb3G18904 gene may play a certain role in the salt tolerance and drought resistance of plants through the signal transduction pathway mediated by ABA,JA,GA,IAA.In order to further understand the function of the Lb3G18904 gene,the recombinant expression vector p CAMBIA3301-35S-GUS-Lb3G18904was constructed,and the p HEC401-CRISPR-Cas9 knockout vector was constructed at the same time.The construction of this part of the vector laid the foundation for further study on its function in salt gland development and stress tolerance of Limonium bicolor in the future.Heterologous transformation of Lb3G18904 in wild-type Arabidopsis thaliana was performed and transgenic T3 homozygous seeds were used to determine its function in salt tolerance.Compared with wild-type Arabidopsis under the same concentration of salt treatment,germination rate,cotyledon growth rate and the root length of Lb3G18904-overexpressed lines were all higher;Less Na~+but more K~+,and the lower ratio of Na~+/K~+was found in Lb3G18904-overexpressed lines;the expression of salt tolerance related genes such as SOS1,NHX1,At GSTU5,At P5CS1 is higher in Lb3G18904-overexpressed lines than in wild type,indicating that Lb3G18904 positively regulates plant salt tolerance.(3)The Lb7G32827 gene is 453 bp and encodes 150 amino acids.It was found that the function of Lb7G32827 is unknown via blasting in NCBI.Bioinformatics analysis results show that Lb7G32827 protein has no transmembrane domain.Subcellular localization experiments show that the protein encoded by Lb7G32827 gene is located in the nucleus.Phylogenetic tree analysis results show that the genetic relationship between Lb7G32827 gene and genes such as walnut and grape are relatively distant.The promoter sequence of Lb7G32827 gene was successfully cloned.Sequence analysis showed that the promoter of Lb7G32827 gene contains not only core promoter elements(TATA-box),common cis-acting elements(CAAT-box),but also hormone-regulated elements such as ABA cis-acting element(ABRE),auxin response elements(TGA-element)and cis-acting elements involved in defense and stress response(TC-rich repeats).According to the analysis of promoter elements,2 abiotic stresses(drought stress,salt stress)and 2 hormones(ABA,IAA)were used to treat seedlings of Limonium bicolor.The results showed that Lb7G32827 gene is involved in salt stress and drought stress response.Moreover,it is induced by ABA and IAA,indicating that the Lb7G32827 gene may play a certain role in the salt tolerance and drought resistance of plants through the signal transduction pathway mediated by ABA and IAA.In order to further understand the function of the Lb7G32827 gene,the recombinant expression vector p CAMBIA3301-35S-GUS-Lb7G32827 was constructed,and the p HEC401-CRISPR-Cas9 knockout vector was constructed at the same time.The construction of this part of the vector laid the foundation for further study on its function in salt gland development and stress tolerance of Limonium bicolor in the future.Heterologous transformation of Lb7G32827 in wild-type Arabidopsis thaliana was performed and transgenic T3 homozygous seeds were used to determine its function in salt tolerance.Compared with wild-type Arabidopsis under the same concentration of salt treatment,germination rate,cotyledon growth rate and the root length of Lb7G32827-overexpressed lines were all higher;Less Na~+but more K~+,and the lower ratio of Na~+/K~+was found in Lb7G32827-overexpressed lines;the expression of salt tolerance related genes such as SOS1,NHX1,At GSTU5,At P5CS1 is higher in Lb7G32827-overexpressed lines than in wild type,indicating that Lb7G32827 positively regulates plant salt tolerance. |
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