Cloning And Functional Verification Of Unknown Genes Lbsgd0090,Lbsgd0130 And Lbsgd1054 Of Limonium Bicolor

Soil salinization has become an increase problem worldwide.It is attracted an increasing attention for scientists to promote green and sustainable economic development by utilizing of saline-alkali land today.Saline soil significantly inhibits plants growth and development with the reduced yield and quality of crops.However,other plant,such as halophyte,can grow normally and complete its life cycles in high-salt environments.Halophytes have developed special salt tolerance mechanism to adapt to high salinity,for example,regulating the expression of related genes.Therefore,it is important to uncover the molecular mechanism of salt tolerance in halophytes,and to improve salt tolerance of crops by gene engineering.Limonium bicolor is a typical recretohalophyte with the structure of salt gland,which can excrete excess salt out of the body,thereby protecting the plant body from salt damage.At present,a large number of physiological studies were carried out on the salt secretion of salt gland in L.bicolor.However,the molecular mechanism of the development of salt gland is still unclear.In addition,many unknown functional genes that related to salt secretion and development of the salt gland were identified in our previous studies.In this study,the role in salt tolerance and salt gland development of the selected three unknown functional genes were to investigate.In this experiment,three unknown functional genes named Lbsgd0090,Lbsgd0130 andLbsgd1054,respectively,were selected to be used for further functional study,they were verified with high expression in the leaves with no epidermal structure differentiation based on the sequencing of the second and third generation transcriptomes of L.bicolor leaves.First,the full-length sequences of three unknown functional genes were cloned and the bioinformatics were performed.To study the function of the genes,the overexpression and mutant strains were obtained by transforming genes into wild-type Arabidopsis thaliana and L.bicolor.Secondly,the phenotypes,physiological and molecular responses to salt stress at germination stage were further verified.The main results are as follows:(1)The Lbsgd0090 encodes 155 amino acids with one transmembrane domain.Subcellular localization of the protein is in nucleus,and it belongs to DUF4216 by BLAST.The protein showed the highest homology with Chenopodium quinoa Willd by performing phylogenetic tree analysis.The regulation domain of GA was found by analyzing the sequence of promoter.The over-expressed homozygous lines were obtained by transforming wild-type Arabidopsis thaliana.The seed germination of the wild-type and over-expressed lines of Arabidopsis thaliana that treated with 0,75,100,125,and 150 mmol L^-1Na Cl,respectively,were performed,the results showed that the physiological indicators,such as germination rate,root length,and fresh weight of the seedlings were decreased with the increase of salt concentration,and there was no significant difference between the wild-type and over-expressed lines at the same concentration of Na Cl treatment.This indicated that the gene might not participate in the regulation of salt tolerance during the seed germination period.(2)The Lbsgd0130 encodes 243 amino acids and has no transmembrane domain.The subcellular localization indicates that the protein is localized in nucleus,and it belongs to the GATA transcription factor by BLAST.The protein showed the highest homology with Spinacia oleracea L.by performing phylogenetic tree analysis.The regulation domain of ABA was found by analyzing the sequence of promoter.The overexpressed homozygous lines were obtained by transforming wild-type Arabidopsis thaliana.The seed germination of the wild-type and over-expressed lines of Arabidopsis thaliana that treated with 0,75,100,125,and 150 mmol L^-1Na Cl,respectively,were performed,the results showed that the physiological indicators,such as germination rate,root length,and fresh weight of the seedlings were decreased with the increase of salt concentration,and there was no significant difference between the wild-type and over-expressed lines at the same concentration of Na Cl treatment.This indicated that the gene might not participate in the regulation of salt tolerance during the seed germination period.(3)The Lbsgd1054 encodes 214 amino acids,and there is a transmembrane dormain.Subcellular localization predicts that the protein is localized in plastids,and it belongs to the LEA2 gene family by BLAST.The protein showed the highest homology with Chenopodium quinoa Willd by performing phylogenetic tree analysis.The regulation domain of ABA and GA were found by analyzing the sequence of promoter.The over-expressed homozygous lines were obtained by transforming wild-type Arabidopsis thaliana.The seed germination of the wild-type and over-expressed lines of Arabidopsis thaliana that treated with 0,75,100,125,and 150mmol L^-1Na Cl,respectively,were performed,the results showed that the germination rate of over-expressed lines under 150 mmol L^-1Na Cl stress was significantly higher than that of wild-type,but there were no significant difference in root length and fresh weight.These results suggested that the over-expressed gene could improve salt tolerance under high(150 mmol L^-1)salt stress at seed germination stage may by regulating the function of ABA and GA.In summary,it was found that the functions of the Lbsgd0090 and Lbsgd0130 genes are notclear.While the over-expressed Lbsgd1054 gene could promote the seed germination rate of Arabidopsis under salt stress.It is speculated that the function performing of the gene is related to the generation of GA and ABA.