Molecular Mechanism Of Larix Olgensis LoHDZ2 In Response To Drought And Salt Stress

HD-Zip family genes can be divided into four subfamilies.Each subfamily has different gene functions and gene diversity,especially playing an important role in plant growth and stress resistance.In order to shorten the breeding cycle of Larch and obtain directional improved materials as soon as possible,the research on growth and stress resistance related genes has important theoretical and research value.In this study,Lo HDZ2 with obvious difference in expression was selected as the follow-up research object combined with the results of q RT-PCR.Firstly,Lo HDZ2 gene was cloned and the gene expression vector was constructed.By stably transforming Larch embryogenic callus and establishing in vitro regeneration system,the transgenic plants of Larix olgensis were obtained,and their growth and resistance were analyzed.Then,transgenic tobacco plants were obtained by leaf disk method for stress resistance analysis.Finally,the stress resistance function of Lo HDZ2 gene was studied by yeast single two hybridization and transcriptome sequencing.The research mainly includes the following aspects:（1）Gene expression analysis of HD-Zip family.According to the early transcriptome sequencing results,13 HD-Zip family genes with complete CDS region were screened and compared by NCBI database,and they were divided into 4 subfamilies according to the conserved domain characteristics of each gene.In order to understand its tissue expression pattern,real-time fluorescence quantitative PCR analysis was used.The results showed that Lo HD-Zip I subclass genes responded to a variety of treatments and had temporal and spatial expression specificity,among which Lo HDZ2 showed obvious expression specificity.（2）Lo HDZ2 gene and promoter analysis.Through transient transformation by tobacco injection,Lo HDZ2 was found to be located in the nucleus and belongs to transcription factor.After comparing the Lo HDZ2 promoter sequence,the element prediction analysis of the gene promoter sequence was carried out.It was found that the promoter sequence not only has the basic transcription elements of the promoter,such as TATA-box,but also ARRLIT,WRKY and other elements related to stress induction.The results of transient genetic transformation of Larch showed that Lo HDZ2 had promoter activity compared with the upstream 1575 bp promoter sequence in the genome database.GUS staining showed that the stem was the deepest blue,and there was a little blue in the leaves and roots,indicating that the gene was mainly expressed in the stem.（3）Acquisition of Larch cell lines transgenic with Lo HDZ2 gene.Through stable genetic transformation of Larch embryogenic callus mediated by Agrobacterium tumefaciens,Lo HDZ2overexpression（Lo HDZ2-OE）and inhibitory expression（Lo HDZ2-RE）transgenic cell lines and regenerated plants were successfully obtained.The analysis of physiological indexes under drought stress showed that the gene could improve the drought resistance of Larch by increasing the content of proline in cells,scavenging reactive oxygen species and enhancing the degree of oxygen resistance of cell membrane;The growth correlation analysis of cell lines showed that the transgenic cell lines grew faster in the early stage,but slowly in the later stage.（4）Abiotic stress analysis of Larch cell lines transgenic with Lo HDZ2 gene.Transcriptome sequencing analysis showed that a large number of genes closely related to abiotic stress were significantly up-regulated in Lo HDZ2 overexpression transgenic cell lines.It is speculated that this gene can improve the drought resistance of plants by regulating downstream and stress resistance related genes.（5）Stable transformation and abiotic stress analysis of tobacco.Lo HDZ2-OE was successfully obtained by stable genetic transformation of tobacco by leaf disk method.The phenotype and physiological indexes of transgenic tobacco under drought stress were measured and analyzed.The transgenic tobacco plants became shorter,the leaves became larger and the flowering time was earlier;Drought resistance analysis showed that the gene could also enhance the antioxidant degree of cell membrane by increasing the content of proline and scavenging reactive oxygen species in transgenic tobacco plants.NBT/DAB staining showed that the staining degree of overexpressed transgenic tobacco was lower than that of wild-type,which further showed that the gene could improve the drought resistance of tobacco.（6）Study on Lo HDZ2 interacting protein and cis acting element.Lo HDZ2 gene has self activating activity and can form homodimer with itself;It interacts with the same subfamily Lo HDZ3 to form heterodimer.Preliminary quantitative results showed that Lo HDZ3 also responded to drought stress and ABA treatment,further indicating that their interaction may improve the drought resistance of larch.It is speculated that HD-Zip I subfamily genes of larch are involved in the regulation of abiotic stress.（7）The cis acting elements that Lo HDZ2 transcription factor may bind were screened by TF centered Y₁H（yeast single hybridization technology centered on transcription factor）,and three known functional elements related to stress resistance were obtained,namely abrelaterd1,WRKY71OS and ABRE.After three repetitions,the three known functional elements were constructed on the p HIS2 vector.After co-transformed with PGADT7-Rec2-Lo HDZ2,the yeast could grow on the defective medium.It is speculated that Lo HDZ2 transcription factor may combine with these three elements to regulate the expression of downstream genes.In conclusion:13 HD-Zip family genes were identified and divided into four subfamilies.Lo HDZ2 gene of Lo HD-Zip I subfamily is involved in regulating the expression of stress resistance related genes,and interacts with Lo HDZ3 gene of the same subfamily to form heterodimer to enhance the stress resistance of Larix olgensis.In this study,the HD-Zip transcription factor family genes of Larix olgensis were identified and studied for the first time,which provided important theoretical basis and gene resources for the analysis of drought stress mechanism and genetic improvement of Larix olgensis.