| Low temperature is an abiotic stress factor that is not conducive to plant growth and production.As the second largest grain crop in China,winter cold in northern China has a negative impact on winter wheat overwintering process.Therefore,the impact of cold climate on winter wheat is the key issue affecting wheat yield.The study of genes related to cold resistance in wheat is helpful to improve the low temperature defense ability of wheat.It can provide important reference value for revealing the regulation mechanism of gene expression and molecular mechanism of cold resistance in wheat,and help to improve crop quality by genetic transformation technology.In this study,the function of promoter and deleted fragment of recrystallization inhibitor IRI gene was verified and analyzed.The main results are as follows:1.The sequence of IRI gene obtained by our team in the early stage is based on the difference of different gene sequences in the IRI gene family.Primer primer design software was used to design specific amplification primers and internal reference primers of IRI gene.Using the specific primers of IRI4,IRI5 and IRI6 as template,real-time fluorescence quantitative PCR was performed to detect the expression activity of IRI4,IRI5 and IRI6 genes in winter wheat M808 under low temperature and methyl jasmonate treatment.The results showed that the expression levels of the three genes were increased under low temperature and methyl jasmonate treatment,and the expression levels of different genes were different in the root,stem and leaf tissues of plants.2.In the early stage,we used the method of chromosome walking to wait for gene IRIp1,combined with BLAST analysis of wheat overlapping group database,and finally amplified six IRI gene sequences with high homology with IRIp1,and selected two of them with high homology.Plant CARE was used to predict cis-acting elements of promoters IRIp1,IRIp2 and IRIp3.The predicted results show that there are some important cis-acting elements in the promoter sequence,such as MYB,MYC,G-box,MEJA responders,light responders,TATA-box and CAAT-box conservative sequences and enhancers.3.Eight GUS fusion expression vectors with different length deletion fragments were constructed from promoters IRIp1,IRIp2 and IRIp3.Agrobacterium-mediated transient transformation of tobacco was used to treat the infected tobacco plants at low temperature and methyl jasmonate.The results showed that the promoter activity of IRIp1 promoter was different under low temperature and hormone stress.The deletion fragments IRIp1-1-GUS,IRIp1-3-GUS and IRIp1-4-GUS had stronger GUS expression activity,and the activity of IRIp1-4-GUS was weaker in response to methyl jasmonate induction at low temperature.The deletion fragments of promoter IRIp2 and IRIp3 were inactive at low temperature and methyl jasmonate treatment,and did not respond to the induction of methyl jasmonate at low temperature.4.To further verify the promoter activity of four deletion fragments of promoter IRIp1under low temperature and methyl jasmonate treatment,eight promoter deletion fragments GUS fusion expression vectors were transferred into Arabidopsis thaliana for genetic transformation experiments.The expression of GUS reporter gene in T2generation of Arabidopsis thaliana seedlings under low temperature and methyl jasmonate treatment was observed by GUS staining.The results showed that the staining of four deleted fragments of IRIp1 in transgenic Arabidopsis thaliana was consistent with that in transient expression experiments. |
