Functional Analysis Of Germin-like Protein Gene TiGLP1 From Thinopyrum Intermedium And Analysis Of TaGLP Gene Family In Wheat

The world’s wheat area and total output ranks first among cereals,and the population that feeds on wheat accounts for more than one third of the world’s total population.At the same time,wheat is also one of the main food crops in China,and wheat production determines national food security.At present,various fungal diseases such as powdery mildew seriously affect wheat yield,so the identification and utilization of disease resistance genes can help to improve wheat disease resistance.When plants are under stress,germin-like genes GLPs play an important role in development,defense and stress.In this study,by analyzing the transcriptome of the previous powdery mildew-induced Trititrigia SN6306,we cloned a germin-like gene TiGLP1 from the powdery mildew resistance related to powdery mildew,and analysed the function of TiGLP1 gene.At the same time,we analyzed and identified the TaGLP gene family in wheat.The main results are as follows:（1）Cloning of TiGLP1 geneThe RNA-seq technique was used to clone and sequence the significantly up-regulated gene TiGLP1 in SN6306 induced by powdery mildew E09.Analysis using online gene sequence analysis software showed that the full-length cDNA of the TiGLP1 gene was 651 bp,and the ORF encoded a protein of 216 amino acids,with a 22 amino acids signal peptide.Alignment and phylogenetic tree analysis of GLPs protein sequences of 14 species showed that the amino acid sequence of TiGLP1 was similar to that of TiGLP2 amplified from Thinopyrum Intermedium.Phylogenetic tree clustering shows that they are closely related.The transcriptome results were verified by powdery mildew infection of SN6306.The results of real-time fluorescence quantitative analysis showed that powdery mildew-induced TiGLP1gene expression was increased.Compared with 0h,the expression level of TiGLP1 gene first increased and then decreased.The expression of TiGLP1 gene reached the peak at 72 h,which was 3.5 times that of 0 h.（2）Preliminary analysis of TiGLP1 functionSubcellular localization shows that it is located outside the cell membrane and is an extracellular secreted protein.Homologous recombination method was used to construct the transformed tobacco overexpression vector pRI101-TiGLP1,and the transgenic tobacco transfected with TiGLP1 gene was obtained by leaf disc transformation.Then T₂ transgenic tobacco was sprayed with Pst DC3000 bacteria,and the leaves after 5 days of treatment were collected for DAB and trypan blue staining to determine the active oxygen in the leaves and observe the dead cells.The results showed that the area of reactive oxygen species（H₂O₂）accumulation and the number of necrotic cells in the transgenic tobacco with TiGLP1 were larger than that of wild-type tobacco.These results indicate that the TiGLP1 gene can enhance tobacco resistance to Pst DC3000.（3）Analysis of TaGLP gene familyA total of 258 wheat GLPs were identified through wheat genome analysis,of which there were 69,86,62,and 41 GLPs lacated on subgenomes A,B,D,and unknown chromosomes.The evolutionary relationship of TaGLP showed that the genes were specific in monocotyledonous and dicotyledonous plants,while TaGLP genes appeared only after monocotyledonous and dicotyledonous plants were differentiated.The conserved motifs and gene structure of TaGLP genes are very different among different subfamilies,but each subfamily has certain similarities.Chinese spring transcriptome analysis showed that 36TaGLP were up regulated during wheat powdery mildew infection,21 of which were located on wheat chromosomes 4A,4B and 4D.This indicates that the fourth homologous group of wheat plays an important role in disease resistance,and qRT-PCR analysis further confirmed their up-regulated expression.