Cloning And Functional Analysis Of Promoter Of Soybean F-box Gene Glyma08g11030 And Identification Of Salt Tolerance Of Rice Germplasm Resources In Seedling Stage

Global climate change intensifies the effects of abiotic stress on plant growth and development.To reveal the molecular mechanism of plant response to abiotic stress and to improve plant stress tolerance by genetic engineering is the main method of crop resistance breeding at present.Promoter,as a switch of gene transcription,plays a "switch" role in regulating gene expression at transcription level.Stress-inducible promoter is a kind of promoter which can only play an active role when plants are affected by abiotic stress and rarely participate in gene transcription.It can regulate the expression of stress-resistant genes in specific sites,periods and environments through external signal and cell signal transduction pathways.The important thing is that it can avoid the negative effects caused by the direct insertion of exogenous stress-resistant genes into plants.It has become one of the most popular applications in plant anti-reverse genetic engineering.The land in the Yellow River Delta region of China is affected by salinization,which results in the decrease of land use efficiency.The study on salt tolerance identification of rice will be expected to improve the land utilization ratio,which is of great significance to the sustainable development of agriculture in China.In this study,the upstream promoter sequence of soybean Glyma08g11030 promoter was cloned,the cis-acting element contained in the promoter was analyzed,and the plant fusion expression vector of promoter-driven GUS gene was constructed.Through transformation of Arabidopsis thaliana and hairy roots of soybean to verify the function of Glyma08g11030 promoter,this study provided stress-inducible promoter for stress-resistant plant genetic engineering.In order to further study the effects of abiotic stress on plant physiology,the salt tolerance of stable lines and some germplasm resources of "Haidao 86" hybrid progenies were identified and analyzed in this paper.The aim of this study was to provide ideal experimental materials for cloning salt-tolerant genes and breeding new salt-tolerant varieties.The main results of this paper are as follows:1.Cloning and cis-acting element Analysis of Soybean F-box Gene Glyma08g11030 PromoterThe promoter of Glyma08g11030 was cloned from soybean cultivar-Williams 82.The sequence of Glyma08911030 promoter included nucleotide sequence of upstream 3000 bp of translation initiation codon ATG.Plantcare predicted cis-acting elements in promoter region online.The results showed that the basic elements of eukaryotes,such as TATA-box,CAAT-box,were found in the promoter region of Glyma08g11030,and there were other regulatory elements,such as cis-acting elements such as ABRE,MBS,Box-W1,HSE,in the promoter region.2.Construction of Plant expression Vector of Soybean F-box Gene Glyma08g11030 PromoterAccording to the position of stress-related cis-acting elements in Glyma8g11030 promoter,the promoter sequence was truncated into three segments.The promoter deletion fragments of 2492 bp,1 185 bp and 342 bp were amplified by PCR.The full-length promoter of Glymag11030 and the binary expression vector of GUS reporter gene driven by different 5’ deletion fragments were constructed.3.Expression Analysis of GUS Gene driven by Glyma08g11030 Promoter of F-box Gene in SoybeanThe full-length promoter plant expression vector was transformed into Arabidopsis thaliana,and the T3 transgenic pure line seeds were obtained.In different tissues of transgenic Arabidopsis thaliana,the full-length promoter of Glyma08g11030 gene drives GUS reporter gene expression in rosette,stem,stem and inflorescence on the 5th,7th day,and in the rosette,stem and inflorescence of Arabidopsis thaliana.Glyma08g11030 promoter was transformed into hairy roots of soybean under 4℃,PEG,ABA and NaCl stress.GUS histochemical staining showed that compared with water-treated hairy roots,in hairy roots treated at 4℃,PEG,ABA and NaCl,there were no significant differences between the two groups.The hairy roots transformed with Glyma08g11030 promoter were stained with GUS.4.Study on Salt Tolerance of "Haidao 86" Hybrid Lines and Germplasm ResourcesThe salt tolerance of 290 hybrid lines and 74 germplasm resources at seedling stage was identified.Treatment with NaCl solution,The salinity is 12‰,and the control was "Haidao 86" and WT.The physiological indexes of 24 selected materials were measured.The results showed that the physiological data of the selected materials were better than those of Yueguang.Finally,15 salt-tolerant and salt-sensitive materials were selected for the identification of salt-tolerant phenotype,the content of MDA and the activity of SOD,POD,CAT enzyme were determined.It was concluded that number 121 and 125 could be used as materials for cloning salt-tolerant genes.