The Function Analysis Of Tomato SlbZIP38 Transcription Factor In Response To Drought And Salt Stresses

Abiotic stress?drought,salt,low temperature,damage and so on?affects the normal growth and development process of plants seriously and even cause plant death.Plants produce a series of complex mechanisms that response and adapt to adversity stress in the process of adapting to the environment.Tomato?Solamum lycopersicum?is one of the widely cultivated important fruits and vegetables.It is also one of the important model plant for plant science research.Tomato is influenced by a variety of abiotic stresses which can reduce the yield in the cultivation process.Therefore,it is of great theoretical significance and practical significance to reveal the resistance mechanisms in tomato.bZIP?basic region/leucine zippermotif,bZIP?is a family of transcription factors that are found in eukaryotes widely.The family members participate in the processes of adversity regulation,plant growth physiological,plant reproduction physiological and biochemical processes,which include plant growth,aging,flower development,damage,fruit mature,mature seeds abiotic stress and so on.Seventy bZIP transcription factors have been predicted in tomato genome.A new bZIP transcription factor,SlbZIP38 gene,was separated and cloned from tomato on the basis of the gene chip and bioinformatics analysis.And it was presumed that it played a role in the abiotic stress process of tomato.Although the bZIP family genes have been studied in many other species,the bZIP family genes in tomato are rarely reported.This paper mainly study the SlbZIP38 gene with plant transgenic technology and molecular biology methods and expound the functional mechanism of SlbZIP38 gene under drought and salt tolerance on the level of phenotype,physiological and molecular.Which can provide the scientific basis to improve the resistance of tomato by genetic engineering techniques.The main results are as followed:?1?Clone the target gene and analysis bioinformaticsThe full CDS of SlbZIP38 gene sequence was cloned by PCR with high fidelity enzyme.According to the analysis of nucleic acid sequence,amino acid sequences,protein homology,promoter regions and subcellular localization,it turns out that SlbZIP38 contains a bZIP domain,a base domain?N-x7-R/K-x9?including 52 amino acids and DOG1?DELAY OF GERMINATION1?domain.SlbZIP38 is clustered to the same branch with 4 genes?ZmbZIP72 from corn,GmbZIP132 and GmbZIP78 from soybean,MsbZIP from alfalfa?responsing to adversity stress.And SlbZIP38 shares high levels similarity of amino acid sequence with ZmbZIP72.The promoter region of SlbZIP38 gene contains elements such as light response element,defence and stress response element,and the SlbZIP38 gene is located in nucleus.It indicates that SlbZIP38 is a typical bZIP family transcription factor and may be in response to adverse responses.?2?The analysis of the induction factorsThe expression levels of the SlbZIP38 gene under exogenous hormones?ABA,GA,Eth,SA,JA?and adversity stresses?salt stress,drought stress,wounding,flooding?were analysised.The results of quantitative PCR shows that the expression of SlbZIP38 gene is induced by abiotice stresses including drought,flooding,temperature,wounding and so on,and that are more significant under the ABA,drougt and salt stress.In addition,the expression levels of SlbZIP38 gene in wild tomato AC⁺⁺and three mature mutants?rin?nor?Nr?are significant different.It is predicted that SlbZIP38gene may be regulated by the ethylene signal pathway and the non-ethylene signal pathway.And the SlbZIP38 gene plays an important role in the process of tomato fruit ripening.?3?Obtain the overexpression and silent transgenic tomatoThe overexpression vector drived by the CaMV35S promoter and CRISPR/Cas9expression editing vectors were constructed and transferred into the wild tomato genome by using the method of agrobacterium mediated.The results of qRT-PCR from the overexpression transformation shows that the expression level of SlbZIP38 gene in the 6 of 14 positive transgenic strains is significantly higher than that in the wild type.Sequencing analysis of CRISPR/Cas9 gene editing mutation sites shows that CRISPR/Cas9 mutation sites in 14 positive plants have been mutanted at A site and two strains are homozygous.While there are 3 lines of B site and the two strains of C are mutant but heterozygous.?4?The sensitivity evaluation to the external ABAThe homozygous overexpression transcription seedings was treated with exogenous ABA?3?M?10?M?to test the sensitiveness under exogenous ABA.It is found that the length of the stem and root of the homozygous overexpression transcription seedings is shorter than WT's under the treatments with exogenous ABA.The results suggest that the homozygous over-expression transcription seedings are more sensitive with ABA treatment than the WT.?5?The evaluation and analysis of drought resistanceThe SlbZIP38 overexpression homozygous lines were used for identifying the drought resistance and determinating the physiological index.The result shows that the drought resistance and the content of MDA,proline and chlorophy?of overexpression transgenic lines decrease.To analysis the level of genes regulated by SlbZIP38 and associated with the signal pathway that the SlbZIP38 gene may involved in,the results shows that ABA synthase gene?SlNCED?and positive regulation genes on the downstream of ABA pathway?SlTAS14?SlAREB1?are expressed at lower levels.SlbZIP38 plays a negative role in the downstream of ABA signal transduction pathway when tomato is under drought stress and reduces the drought resistance of tomato.?6?The evaluation and analysis of salt resistanceThe SlbZIP38 overexpression homozygous lines were used for identifying the salt resistance and determinating the physiological index.The result shows that the salt resistance and the content of MDA,proline and chlorophy?of overexpression transgenic lines decrease compared to that of wild tomato after the treatment of400mM NaCl for five days.To analysis the level of genes regulated by SlbZIP38 and associated with the signal pathway that the SlbZIP38 gene may involved in,the results shows that only the SlAREB1 is consistent with drought stress and has the same expression trend of the four marker genes in the ABA pathway?SlPP2C2?SlNCED?SlTAS14?SlAREB1?.The difference is the expression levels of SlPP2C2?SlNCED and SlTAS14 increase and the overexpression plants increase more than that of wild type.Hence,the mechanism of SlbZIP38 under salt stress is not exactly consistent with drought stress.The result of RNA-seqencing between the OE lines and WT showes that a total of99 different expression genes are found,and 43 genes level are up regulation and 56genes are down regulation.An analysis was conducted that SlbZIP38 gene respond to high salt stress by down-regulating proline enrichment protein genes and protein inhibitor genes.?7?The analysis of proteins interacting with SlbZIP38To understand the mechanism of SlbZIP38 on the protein level,this study screened the tomato cDNA library by the Matchmaker Gold Yeast Two-Hybrid?Y2H?system to find out proteins interacting with SlbZIP38 protein in a tomato.Twenty-five interaction proteins were identified,and the functions of the interaction proteins were stress related protein genes,pathogen defense genes,ubiquitin pathway related genes,growth regulating genes in a pathway,sugar accumulation genes,mature fruit genes and so on.