Spinach SpoPH1 Gene Cloning And Function Analysis Of High Temperature Response

Spinach(Spinacia oleracea L.)is one of the important economic green leafy vegetables in China.It has rich nutritional value and is widely grown in the world and in China.It has the characteristics of cold tolerance but not heat resistance.High temperature stress seriously affects the growth and development of spinach,In this paper,heat-resistant spinach Sp75 and heat-sensitive spinach Sp73 are used as research materials.The molecular mechanism of spinach Spo PH1 participating in the response to high temperature stress is studied using molecular biology methods.PH domain(Pleckstrin homology domain),a functional region consisting of 100-120 amino acids,is a functional region that exists in a variety of signaling proteins and cytoskeleton-related proteins,and is involved in cell signaling and cytoskeleton formation,membrane transport,etc.Studying the regulation of Spo PH1 on high temperature response will provide important reference for further analysis of the molecular mechanism of high temperature response in plants.1.Analysis of the spinach genome database found that there are two genes encoding PH protein in the spinach genome.Using molecular PCR technology,the Spo PH1 gene was cloned,with a maximum open reading frame of 450 bp,encoding a total of 150 amino acids,and a theoretical molecular mass of 17.4 k Da.2.Using homologous sequence alignment analysis,it was found that the Spo PH protein has high homology with the PH protein of many plants and contains a more conserved PH domain,indicating that the PH domain is more conservative in evolution.3.Real-time fluorescence quantitative RT-q PCR analysis of Spo PH1 expression pattern.After high temperature treatment at 37°C,the expression of Spo PH1 gene in the spinach heat-resistant material Sp75 showed an overall upward trend with the extension of treatment time.However,the spinach heat-sensitive material Sp73 showed an overall downward trend,These results indicate that the response pattern of Spo PH1 gene in Sp75 and Sp73 is different under high temperature stress.The expression of Spo PH1 gene in different organs was detected at the six-leaf stage and the bolting stage of spinach.The results showed that at the six-leaf stage,Spo PH1 gene had the highest gene expression in lateral roots,the lowest gene expression in main roots,and the expression level of cotyledons in leaves was higher than that of three pairs of true leaves.In the bolting stage,the expression of Spo PH1 gene was highest in lateral roots and lowest in leaves.4.The p BI121-Spo PH1-GFP plant expression vector was constructed and introduced into(Nicotiana tabacum)leaf cells for transient expression analysis of tobacco.The results showed that the Spo PH1 protein was localized to the plasma.5.By constructing p YES2-Spo PH1 yeast expression vector and transducing into a wild-type yeast,Spo PH1 transgenic yeast was obtained and subjected to high-temperature phenotypic analysis.The results showed that compared with wild-type yeast strains,Spo PH1 transgenic yeast was more resistant to high temperature compared with wild-type yeast strains at 37°C,which indicated that Spo PH1 gene played a positive regulatory role in yeast cells under high temperature stress.6.By constructing p ET32a-Spo PH1 prokaryotic expression vector,the His-Spo PH1 fusion protein was induced and purified.The protein purity was verified by western blotting,and the in vitro binding experiments of phosphatidylinositol confirmed that the Spo PH1 protein had binding to phosphatidylinositol.7.The p BI121-Spo PH1 plant expression vector was constructed and Spo PH1 overexpression Arabidopsis plants were obtained by Agrobacterium infection method.Spo PH1 mutant back-up strains were obtained by Agrobacterium infection.Root phenotypic analysis showed that compared with wild-type Arabidopsis,Spo PH1 overexpressed Arabidopsis had the longest root length and At PH1 Arabidopsis mutant had the shortest root length,The root length of Spo PH1 homozygous Arabidopsis mutant back-to-back strains had a certain recovery compared to the mutant root length,which proved that Spo PH1 may affect the growth of Arabidopsis roots.