Functional Analysis Of NtMLP423 Gene In Tobacco Under Drought And Chilling Stress

Tobacco is an important economic crop,and also a significant scientific research model plant.In the process of its growth and development,it often suffers from a variety of abiotic stresses,among which drought and chilling stress have become the main environmental factors restricting the cultivation and quality of tobacco.It is of great theoretical significance for breeding and cultivating resistance varieties by biological technology to study the molecular mechanism of tobacco resistance,excavate resistance genes and explore the gene function.The major latex protein(MLP)is plant specific protein,and belongs to the second largest subfamily of the Bet v 1 family.In this study,the major latex protein NtMLP423 gene was cloned from tobacco.We constructed overexpression and antisense vectors,and obtained transgenic tobacco.Then we further explored the function of transgenic tobacco under drought and chilling stress.The main results are as follows:(1)The bioinformatics analysis of NtMLP423 found that the NtMLP423 had a conserved glycine-rich loop.The three-dimensional structure was composed of seven ? sheets and two ? helices,which could form a Y-shaped hydrophobic cavity.The hydrophobic cavity can combine with different ligands to perform different biological functions.(2)The expression analysis of NtMLP423 under hormone induction and abiotic stress showed that NtMLP423 gene was induced by a variety of hormones.Drought,oxidation and chilling stress could induce the expression of NtMLP423 gene,suggesting that NtMLP423 gene may play a role in abiotic stress.(3)The wild-type and transgenic tobacco were treated with drought stress,and the results showed that overexpression of NtMLP423 increased the drought resistance of tobacco.The survival rate of overexpression plants was significantly higher than that of wild-type and antisense plants,and the degree of wilting was just the opposite under drought stress.The physiological indicators were measured under drought stress,and the results showed that the relative water content of overexpression plants was higher and the water loss rate was lower under drought stress.Proline content of overexpression plants were significantly higher than that of wild-type and antisense plants,and malondialdehyde and relative electrical conductivity were lower than that of wild-type and antisense plants,which indicated that overexpression plants improved osmotic adjustment ability and reduced the degree of membrane damage under drought stress.The measurement results of photosynthesis related indicators showed that overexpression of NtMLP423 maintained photosynthetic capacity under drought stress.At the same time,overexpression of NtMLP423 increased the activity of antioxidant enzymes and decreased the accumulation of reactive oxygen species.(4)The transgenic and wild-type tobacco were treated with ABA and the results showed that the overexpression plants promoted the stomata closure and reduced the seeds germination rate under ABA treatment.Overexpression of NtMLP423 increased the ABA content under drought stress.The ABA synthesis and decomposition related genes were analyzed by q RT-PCR,and the results showed that overexpression of NtMLP423 increased the expression of ABA synthesis related genes.In addition,yeast two-hybrid and Bi FC experiments proved that NtMLP423 could interact with Nt ABI5.These results indicated that overexpression of NtMLP423 could improve plant resistance to drought stress through the ABA pathway.(5)In order to explore the transcriptional regulation mechanism of NtMLP423 under drought stress,we screened the Nt WRKY71 transcription factor.The expression trends of Nt WRKY71 and NtMLP423 was consistent under drought stress.Luciferase,yeast one-hybrid and EMSA experiments proved that Nt WRKY71 could directly bind to the NtMLP423 promoter through the W-box site and activate its transcription.Nt WRKY71 was silenced by VIGS technology,and the results showed that the expression of Nt WRKY71 and NtMLP423 gene was significantly reduced in silencing plants.At the same time,the wilting of the silencing plants was more serious than that of the control group under drought stress.The results showed that silencing Nt WRKY71 reduced the resistance to drought stress.In addition,the expression levels of drought stress related genes in silencing plants were also lower than those in the control group.These results indicated that the transcription factor Nt WRKY71 played a positive role in the regulation of drought stress by NtMLP423.(6)Wild-type and transgenic tobacco were treated with chilling stress and the results showed that overexpression of NtMLP423 increased tobacco resistance to chilling stress.Compared with wild-type and antisense plants,the germination rate of overexpressing NtMLP423 plants was significantly increased,the degree of wilting was reduced,and the chlorosis was relatively mild under chilling stress.At the same time,the proline and soluble sugar content of the overexpression plant was higher than that of the wild-type,while the malondialdehyde and relative electrical conductivity were significantly reduced under chilling stress.This indicated that overexpression of NtMLP423 enhanced the osmotic adjustment ability and reduced membrane damage of the transgenic plants.Meanwhile,the net photosynthetic rate,Fv/Fm and chlorophyll content of overexpression plants were significantly higher than those of wild-type and antisense plants,which maintained the photosynthetic capacity under chilling stress.Overexpression of NtMLP423 improved the activities of antioxidant enzymes and decreased the accumulation of reactive oxygen species under chilling stress.Overexpression of NtMLP423 increased the expression levels of antioxidant enzyme genes and cold stress related genes under chilling stress.(7)The transcriptome analysis was performed on the wild-type and overexpression lines after chilling treatment,and the transcription factors with significant differences were screened out from the transcriptome data.The results of q RT-PCR analysis showed that the expression trends of NtMYB108 and NtMLP423 were consistent under chilling stress.Luciferase,yeast one-hybrid and EMSA experiments proved that NtMYB108 could bind to the promoter of NtMLP423 through TAACTG motif and activate the transcription of NtMLP423.The role of NtMYB108 transcription factor in regulating chilling resistance of NtMLP423 was investigated by VIGS.The results showed that silencing NtMYB108 plants significantly reduced the expression of NtMYB108 and NtMLP423.The silencing plants were treated with chilling stress,and it was found that the silencing plants reduced the tolerance to chilling stress,and the degree of wilting was more serious.q RT-PCR analysis showed that the expression level of cold resistance related genes of silencing plants was lower than that of the control group under chilling stress.These results indicated that transcription factor NtMYB108 played a positive role in the regulation of chilling stress by NtMLP423.