Clone And Functional Analying Of Salt-tolerant Genes MDH And BADH In Spartina Alterniflora

With the rapid development of the global economy and the rapid growth of population,human beings urgently need more land to meet the needs of production and life.Land salinization has become an important abiotic stress factor restricting agricultural development because of unreasonable use of land by humans.High salt and drought stress could damage the integrity of the plant membrane system,destroy the structure and function of chloroplasts and also could inhibit the activity of enzymes,etc.The cloning salt-tolerant genes from salt-tolerant plants by transgenic technology and cultivating transgenic crops or plants could increase crop yield and utilization of saline-alkali land and arid land.Spartina alterniflora is belong to gramineae recretohalophyte in plant taxonomy,and is suitable for growing in the intertidal zone.There are salt glands on the leaves of Spartina alterniflora.It has certain ability to resist wind and salt stress.It is a very good plant material for salt tolerance gene mining.In this study,we firstly cloned monodehydroascorbate reductase gene(SaMDH1 and SaMDH2)and bataine aldehyde dehydrogenase gene(SaBADH)from Spartina alterniflora.Secondly,we investigated the expression profiles of SaMDH1?SaMDH2 and SaBADH gene under 600 mmol/L NaCl stress and 20% PEG stress conditions in Spartina alterniflora.Finally,we constructed the overexpression vector of SaMDH1?SaMDH2 and SaBADH gene which were successfully transformed into Arabidopsis thaliana.Then we studied the salt tolerance of 15 homozygous transgenic Arabidopsis thaliana under salt stress conditions.The relevant experimental results are as follows:(1)The cloning and sequence analysis of SaMDH1?SaMDH2 and SaBADHWith the help of Spartina alterniflora transcriptome sequencing result,we cloned the full-length cDNA sequence of MDH and BADH gene from Spartina alterniflora by RACE strategy and then named SaMDH1?SaMDH2 and SaBADH respectively.Bioinformatic analysis showed that the full-length cDNA of SaMDH1 gene was 1 832 bp and the open reading frame encoded 476 amino acids;the full-length cDNA of SaMDH2 gene was 1 978 bp and the open reading frame encoded 494 amino acids;the full-length cDNA of SaBADH gene was 1 983 bp and the open reading frame encoded 504 amino acids.N-J phylogenetic tree showed that the MDH of Spartina alterniflora and the MDH of Eleusine coracana had high homology;the BADH of Spartina alterniflora and the BADH of Zoysia tenuifolia had high homology.We analyzed that SaMDH1?SaMDH2and SaBADH were localized to peroxisomes?chloroplasts and chloroplasts respectively from N-J phylogenetic tree.(2)The expression profiling of SaMDH1?SaMDH2 and SaBADHWe studied the expression change of MDH1?MDH2 and BADH gene in Spartina alterniflora under different stress times of 600 mmol/L NaCl and 20% PEG by qRT-PCR technology.The results showed that the expression of SaMDH1?SaMDH2 and SaBADH gene increased first and then decreased with the prolongation of treatment time,and the difference reached a significant level compared with the control.This indicated that SaMDH1?SaMDH2 and SaBADH gene were induced by NaCl and PEG and they were closely related to the salt tolerance and drought tolerance of Spartina alterniflora.(3)The studies of heterologous overexpressed SaMDH and SaBADHWe screened out the homozygous transgenic Arabidopsis thaliana through the marker hygromycin gene.Then we analyzed the expression of foreign genes by qRT-PCR technology and selected two lines with higher expression levels for salt tolerance analysis with wild type.The results showed that homozygous transgenic Arabidopsis thaliana had more growth advantages than the wild type in the three aspects of bending roots and seed germination and physiological and biochemical indexes of the leaves.This indicated that overexpression of SaMDH and SaBADH gene could improve the salt tolerance of the model plant Arabidopsis thaliana.