Cloning And Study On Unknown Functional Genes Lbsgd0089,Lbsgd0109 And Lbsgd0119 Of Limonium Bicolor L.

With the increase of soil salinization,hundreds of millions of hectares of land around the world are affected by salinization.Therefore,the salt tolerance of crops grown on saline-alkali soils is becoming increasingly important.At present,saline-alkali land can be improved and utalized by planting halophytes.It is urgent to thoroughly study the salt-tolerance mechanism of halophytes and the function of relevant salt-tolerance genes,this is not only conducive to the improvement of saline-alkali land by halophytes,but also can transfer salt-tolerant genes into non-halo-phyte through transgenic technology,thereby enhancing its salt tolerance.As a exo-recretohalpphytes,Limonium bicolor can excrete excess salt ions to the outside through the epidermal salt gland structure(salt gland),thereby avoiding salt damage.At present,there are few studies on the molecular mechanism of the salt secretion and development of L.bicolor salt gland.In our previous studies,a large number of unknown functional genes related to salt secretion and development of the salt gland was identified.However,thier functions are unclear.Therefore,this article is dedicated to the research of three unknown functional genes.According to the sequencing results of three generations of transcriptomes from the early stages of the salt gland development of L.bicolor,three unknown functional genes Lbsgd0089,Lbsgd0109 and Lbsgd0119 were highly expressed in the early stages of salt gland development.In the present thesis the full lengths of these genes were cloned and the bioinformatics analysis was performed.Overexpression vectors and knockout vectors were constructed to transform wild-type Arabidopsis thaliana and L.bicolor,and the phenotypes and functional analysis of transgenic plants were carried out;at the same time,transient expression in tobacco was performed and subcellular localization was done.The main results are as follows:(1)Three unknown functional genes Lbsgd0089,Lbsgd0109 and Lbsgd0119 were screened to look insight into thier roles in plant growth,development and adaptation to abiotic stress based on the sequencing results of the three-generation transcriptome of L.bicolor.First,full-length cloning and bioinformatics analysis of three genes was performed.Lbsgd0089 gene has a base sequence of 1212 bp and encodes 403 amino acids.According to the blast results,the species with the highest homology is Punica granatum L.and possesses the DUF707 superfamily domain but unknown.Lbsgd0109 gene has a base sequence of 1260 bp and encodes419 amino acids.Phylogenetic tree analysis shows that Lbsgd0109 has a relatively high homology with Nepenthes and has two unknown functional domains,DUF4409 and DUF239.Lbsgd0119 has 516 bp and encodes 171 amino acids.According to the phylogenetic tree,the gene has the highest homology with the histone H1 of Phalaenopsis,and five transmembrane regions were found in the transmembrane domain analysis.It is a transmembrane protein and may be related to the respones of plant to environmetal stress.(2)The expression vectors of Lbsgd0089,Lbsgd0109 and Lbsgd0119 were constructed by homologous recombination,and the resulting homologous products were transformed into E.coli and Agrobacterium in turn,wild-type Arabidopsis was transformed,and pure overexpression lines have been obtained.The relative expression was analyzed by quantitative real-time PCR.Differently expressed lines for Lbsgd0089(89-OE6,89-OE1,and 89-OE3),Lbsgd0109(109-OE4,109-OE8,and 109-OE2)and Lbsgd0119(119-OE6,119-OE3 and 119-OE2)were used to further study their roles.Phenotypic observation and functional analysis were carried out on three overexpressed strains of three genes.(3)The seeds of wild-type Arabidopsis thaliana,over-expressing lines of Lbsgd0089,Lbsgd0109 and Lbsgd0119 genes were treated with 0 and 100 m M Na Cl,respectively.The results showed that under Na Cl stress,the seed germiantion parameters(germination rate,germination index and cotyledon growth rate)of both wild-type and Lbsgd0089,Lbsgd0109 and Lbsgd0119 over-expressing Arabidopsis lines significantly decreased.However,under the same salt concentration the over-expressing lines showed significantly lower germiantion parameters than wild type and no significant difference among the different over-expressing lines under Na Cl stress,indicating that the three genes had negative effect on the regulation of salt tolerance at the germination stage.In order to further explore the role of the Lbsgd0089,Lbsgd0109 and Lbsgd0119 genes in the mechanism of salt resistance,we measured the expression levels of the salt-tolerance related marker genes such as SOS1,HKT1,NHX1,P5CS1 of each over-expressing lines and wild type.The results showed that the expression levels of the marker genes of the three genes over-expressing lines are all lower than those of the wild type,indicating that the unknown genes-reduced salt tolerance is via downreguanlation of SOS1,HKT1,NHX1,P5CS1.(4)Transient expression of the Lbsgd0089,Lbsgd0109,and Lbsgd0119 genes were subcellularly localized using tobacco epidermis.It was found that Lbsgd0089 was expressed in the nucleus,so we speculate that this gene may be a transcription factor or related to some functions of ribosomes;and Lbsgd0109 and Lbsgd0119 were expressed in the cytoplasmic membrane,it is suspected that it may play an important role in receptor recognition and signaling.The key role that these three genes play in the development of L.bicolor salt gland needs further investigation.