Cloning And Preliminary Functional Analysis Of GDSL Lipase Gene CpGLIP1 Promoter In Chimonanthus Praecox(L.)

Wintersweet is a deciduous bush of the genus Chimonanthus praecox,which is wellknown in my country.The wax plum has the ability of resistance to cold,drought and pruning,which is related to the regulation mode of genes that resist stress.GDSL is a class of lipolytic enzymes.In previous studies,it was found that this enzyme can play a role in lipid metabolism,organ development and stress.In the previous experiments,a GDSL enzyme gene of the wintersweet was found and named CpGLIP1,it was found that it can respond to low temperature and drought stress,and at the same time can be induced by some plant hormones JA and GA.It is speculated that CpGLIP1 may play a role in various abiotic stress responses of plants.On this basis,the function verification of the poplar CpGLIP1 gene of the transgenic wax plum obtained from the previous research in the laboratory was carried out under drought and low temperature stress,and the gene promoter was cloned and preliminarily analyzed to further verify the gene.To explore the expression and regulation mode of CpGLIP1 gene,and to study the molecular texture of the gene in the stress of the wintersweet.The main findings show that:1.Cloning of CpGLIP1 Gene Promoter SequenceIn this study,the promoter sequence of CpGLIP1 gene with a length of 2078 bp was successfully cloned from the genomic DNA of the wintersweet by conventional PCR method,which was named CpGLIP1pro.Through bioinformatics analysis,the promoter sequence contains LTR,STRE,MYC and MYB related to plant abiotic stress,as well as hormone-induced related CGTCA-motif,ABRE elements,etc.It is speculated that the promoter of CpGLIP1 gene can respond to a variety of abiotic stress and hormonal induction.2.Cloning of CpGLIP1 gene promoter deletion sequence,construction of vector and stable expressionThe elements of the promoter sequence of CpGLIP1 gene were analyzed and primers were designed.A deletion fragment CpGLIP1pro-P1 was obtained by PCR technology,and it was connected with the expression vector pBI121 containing the GUS gene to obtain the expression vector pBI121-CpGLIP1pro/CpGLIP1pro-P1.Arabidopsis thaliana was stably expressed by Agrobacterium-mediated transformation,and the transgenic Arabidopsis thaliana T2 generation line was screened by karamycin(Kan),which was subjected to GUS histochemical staining and PCR molecular verification.3.Tissue-specific analysis of the CpGLIP1 gene promoterThe germination period of transgenic Arabidopsis thaliana seeds with CpGLIP1pro and CpGLIP1pro-P1 sequences was histochemically stained with various organs of mature plants.The results showed that the seeds were only stained at the base of the cotyledons during the germination period,and the leaves could be partially stained.The stems,flower bases,and filaments of mature plants can be dyed.The results showed that both CpGLIP 1pro and CpGLIP1pro-P1 could drive the expression of GUS gene in different growth stages of Arabidopsis thaliana,but with certain tissue specificity.4.Analysis of CpGLIP1 Gene Promoter Stress ResponseThe transgenic CpGLIP1pro and CpGLIP1pro-P1 Arabidopsis were induced by ABA,GA and JA hormones,low temperature at 4℃,simulated drought with PEG6000,and after dark treatment,the GUS gene and GUS enzyme activity of the transgenic Arabidopsis were quantitatively analyzed.The results was shown that under the drive of CpGLIP 1pro and CpGLIP1pro-P1 promoters,GUS gene responded to GA,JA,4℃.PEG6000 and dark treatment,but less to ABA hormone.5.Functional verification of transgenic CpGLIP1 gene in poplarAccording to the CpGLIP1 gene transfected INRA 717-1B4 poplar in the laboratory’s previous research,the genome PCR was carried out,and after combining with the fluorescence quantitative detection in the previous experiment,three lines with different expression levels(OE1,OE2,OE4)were selected for tissue culture expansion,and finally several lines were obtained.CpGLIP1 gene poplar was subjected to abiotic stress experiments.In vitro leaf water loss experiment showed that the CpGLIP1 gene poplar leaf water loss rate was lower than that of the wild type.Stomatal observation and induced stomatal closure experiments also showed significant differences between transgenic and non-transgenic.After the PEG6000 simulated drought experiment,the electrical conductivity,relative water content,SOD enzyme activity,net photosynthetic rate and intercellular CO2 concentration of the transgenic CpGLIP1 gene poplar and wild type were detected.The results showed that the transgenic CpGLIP1 gene poplar has strong drought resistance ability than in wild-type poplars.Through the low temperature stress experiment at 4℃,the electrical conductivity,proline,chlorophyll content,SOD enzymes,net photosynthetic rate and intercellular CO2 concentration of the transgenic poplars were detected,and most of the results showed that the transgenic poplars were resistant to low temperature and stronger than wild-type poplar.It was preliminarily concluded that transgenic poplars with CpGLIP1 enhanced the effect of plants in drought and cold resistance.