Study On Regulation Mechanism Of MtRAVs On Growth And Adversity Stress Responses In Medicago Truncatula

As a kind of leguminous forage grass,alfalfa is the most important economic crop in animal husbandry because of its strong adaptability,high yield,good quality and high nutritional value.However,due to the limited area of high-quality cultivated land in China,alfalfa can only be promoted to a limited extent in order to ensure food security.In order to expand the planting area and improve the yield of alfalfa,it is imperative to select some late maturing alfalfa varieties with strong stress resistance.Using molecular biological methods to study the function and mechanism of alfalfa related genes,which promote late ripening,and resist environmental stress,these results can not only improve the basic research,but also provide the basis for the cultivation of new varieties.The purpose of this study was to isolate MtRAVs genes from Medicago truncatula,and study these genes,identify their functional differences,and analyze their regulatory mechanism.RAV（Related to ABI3/VP1）transcription factors belong to AP2 or B3 superfamily and are plant specific transcription factors.Previous studies have shown that salt,polyethylene glycol（PEG）,low temperature and drought can induce the expression of RA Vs genes in plants,and then enhance the resistance of plants to stress（salt,drought and low temperature）through the activation of stress resistance related genes;the molecular regulation of plant stress resistance by RAV may be based on DREB/CBF regulation pathway,or independent on it,and completed by other regulatory mechanisms.At the same time,the expression of RAV is also related to the hormone level in plants,and participates in the signal transduction of some plant hormones;some studies have shown that RAV inhibits the expression of flowering gene FT,thus delaying the flowering time.Although many studies have confirmed that the function of RAV is related to plant growth,development and stress resistance,the regulatory mechanism involved in RAV is not clear,most of which are conjectures,and there is no direct evidence to confirm it.In addition,the functional redundancy and differences among members of RAV family should be further explored.In our previous transcriptome sequencing study,we found that under salt stress,the expression of some RAVs genes transcripts changed.In this context,the purpose of this paper is to isolate and identify MtRA Vs genes from Medicago truncatula,study their functions and analyze their differences.Through protein screening and high-throughput sequencing,we can understand the molecular mechanism of MtRAVs involved in growth,development and stress resistance regulation,and lay a foundation for further research.This study focuses on MtRAVs gene cloning,bioinformatics analysis,gene expression pattern,subcellular localization,physiological,biochemical analysis and RNA-Seq sequencing analysis of MtRAVs transgenic Medicago truncatula,and the following results are obtained:1.According to bioinformatics analysis,three RAV genes were cloned from M.truncatula,MTR₁g093600,MTR₅g053920 and MTR₁g116920,and named as MtRAV1,MtRAV2 and MtRAV3.The molecular structure characteristics and sequence homology of three MtRAVs genes were analyzed,and it was found that MtRAV1,MtRAV2,and MtRAV3 encode 384,378,and 298 amino acids,respectively,which are closely related to the RAV family proteins of legumes.The upstream promoter sequences of the three genes coding regions were analyzed respectively,and these promoter contains a variety of cis-acting elements related to photosynthesis,hormones and stress.The similarities and differences of molecular structure,gene location and cis acting elements in promoter among MtRAVs suggested that there was functional redundancy and differentiation among three MtRAVs genes.2.The qRT-PCR results show that MtRAVs expressed in flowers,leaves,stems and roots,MtRAV1 has a high expression level in leaves,MtRAV2 has a high expression level in roots,and MtRAV3 has a high expression level in flower organs.The expression level of MtRAV1 was the highest in the whole plant of Medicago truncatula,and that of MtRA V3 was the lowest.Salt stress can induce the expression of MtRA Vs gene at different degrees,and the transcription level is rapidly up-regulated within 1h,and then the expression is gradually down-regulated with the extension of stress treatment time.MtRAVs gene also responded to PEG osmotic stress to varying degrees,but the transcriptional level of MtRAV3 was upregulated much larger than that of MtRAV1 and MtRAV2.The expression of MtRAV3 gene was significantly induced by low temperature,while the transcription level of MtRAV1 and MtRAV2 was induced with a small degree.Gibberellin GA3,abscisic acid ABA and salicylic acid SA had different effects on MtRAVs gene expression.At the same time,the infection of Fusarium spp.caused upregulation of the expression of MtRAVs.3.The 35S::MtRAVs-GFP fusions were transformed into protoplasts of M.truncatula mesophyll cells,and the transformed protoplasts were monitored by confocal laser scanning microscope.The results revealed that M.truncatula RAV fusion proteins all localized in the cell nucleus.4.Transgenic Arabidopsis lines were successfully obtained by inflorescenc infection.The results showed that the number of primary and secondary branches in transgenic Arabidopsis was significantly higher than that of wild type,but there was no significant difference for tertiary branch between them.The total number of branches in MtRAVs transgenic Arabidopsis was also significantly higher than that of wild type.The overexpression of MtRAVs enhanced the resistance of Arabidopsis to salt stress and osmotic stress.The results of stress experiments in 200 mM NaCl and 500 mM mannitol showed that transgenic Arabidopsis seedlings and wild type showed obvious difference in resistance.Transgenic Arabidopsis showed strong tolerance to stress,and phenotype indexes such as root length,fresh weight and survival rate were significantly better than those of wild type.At the same time,the overexpression of MtRAVs increased the transcription level of genes related to abiotic stress in Arabidopsis thaliana,and MtRA Vs overexpression weakened the sensitivity of Arabidopsis thaliana to exogenous ABA.In addition,MtRAV3 overexpressionenhanced the tolerance of Arabidopsis to freeze injury.These results indicated that the resistance of MtRAVs transgenic plants to osmotic stress,salt stress and freeze injury washigher than that of wild type plants.5.The overexpression of MtRAVs resulted in obvious dwarfing and late flowering of M.truncatula.The effect of MtRAV3 on plant dwarfing and delayed flowering was significantly weaker than that of the other two genes in the RAV family.At the same time,the overexpression of MtRAVs also led to the decrease of leaf and floral organs and the increase of branches.The results of scanning electron microscopy showed that the mesophyll cells of transgenic plants were significantly smaller than those of wild type plants.Fluorescence quantitative analysis of transgenic plants showed that the overexpression of MtRAVs significantly inhibited the expression of the key genes involved in the synthesis of strigolactone.MtRAV3 transgenic M.truncatula showed enhanced resistance to abiotic stresses（salt,osmotic,drought）.In addition,the overexpression of MtRAVs significantly inhibited the expression of GA3ox1,which is the key enzyme of GA4 synthesis,and the level of GA4 hormone decreased.Moreover,ga3oxl mutant showed the same phenotype as MtRAVs transgenic plants.6.In order to analyze the regulatory mechanism of MtRAVs on the growth and development of M.truncatula,MtRAV1/2/3 was used as bait protein to screen the cDNA library of M.truncatula by yeast two hybrid system,found the possible interaction proteins of MtRAVs.7.RNA-seq analysis of transgenic M.truncatula showed that the overexpression of MtRAVl and MtRAV2 increased the expression levels of transcripts related to hormone synthesis,signal transduction and transcription factor regulation,but the expression of thylakoid,photosynthetic system and oxidoreductase complex were inhibited;MtRAV3 positively regulated heme binding and tetrapyrrole binding and the expression of iron binding transcripts.These results indicate that MtRAVs directly regulate the hormone levels related to plant growth and development,and affect the operation of photosynthetic system.At the same time,MtRAV3 also plays a role in oxidative stress through the regulation of tetrapyrrole compounds.These results suggest that MtRAVs are important transcription factors regulating plant growth and development,and play a role in regulating plant stress tolerance and other aspects.In this study,the fuction of MtRAVs in plant growth,development and stress resistance regulation was preliminarily analyzed,which provided a theoretical basis for the breeding of stress resistant and late maturing alfalfa varieties.