| Spinach(Spinacia oleracea L.)Sp75 and Sp73 are spinach inbred lines independently bred by Shanghai Normal University.Sp75 was selected as the material for spinach genome sequencing because of its strong resistance to downy mildew,heat resistance,fast growth,and high purity.In recent years,the progress in the study of spinach genome and molecular theory of important traits has facilitated the study of the related mechanisms of spinach's heat resistance and disease resistance at the level of molecular biology and molecular breeding.In the early stage of the laboratory,aiming at the difficulty of over-summer cultivation of spinach in Shanghai,we carried out the breeding of new heat-resistant spinach varieties,the cloning of heat-resistant genes,and the development of molecular markers,in order to solve the production problems of Shanghai spinach over-summer cultivation difficulties.an effective plan.The use of genetic engineering technology to carry out research on transgenic spinach is also an effective way to cultivate new varieties of spinach that are resistant to stress and disease.In this study,spinach Sp75 and Sp73 were selected as the research objects,and the medium and culture conditions of the regeneration system of spinach Sp75 and Sp73 were optimized by screening the primary medium for seed germination and seedling growth,adjusting the type and concentration of hormones,and selecting culture conditions.,laying a theoretical foundation for further genetic transformation of spinach Sp75 and Sp73.At the same time,based on the screening of yeast two-hybrid library with So Hsf A1 d as bait,a total of 125 positive clones were obtained for sequencing and identification,and a total of 27 candidate genes were expressed,of which 8 were candidates for heat resistance-related proteins.q RT-PCR to analyze the expression pattern of the candidate protein genes,to lay a foundation for analyzing the regulation mechanism of spinach HSFA1 d in spinach heat resistance.The main findings are as follows:1.The seed germination experiment of Sp73 showed that the seed germination of Sp73 was extremely sensitive to water,and reducing the humidity of the medium was beneficial to seed germination.The control increased by 16%;the elongation and growth of the radicle after germination of Sp73 seeds still needs to grow under the condition of low relative humidity.The comparative experiment shows that the use of moist filter paper can promote the development of radicle after germination,and then it can be the same as Sp75.Grow on MS minimal medium;2.The optimization results of Sp75 tissue culture showed that the callus induction rate of explants under 6-BA + 2,4-D was 13.5% higher than that of the 6-BA + NAA combination,but the size was lower than that of the latter.The size is 0.4 levels lower than the latter.The comprehensive comparison showed that 6-BA + 2,4-D was beneficial to callus induction from leaves,and the optimum concentration was 1 mg/L6-BA + 1 mg/L 2,4-D;+ NAA is more suitable for callus induction from petiole,and the optimum concentration is 2 mg/L 6-BA+0.8 mg/L NAA;Under the condition of BA + 0.4 mg/L NAA,the induction rate of adventitious buds was the highest,but the induction rate was only 2%;the induction rate of callus from sprouts was 37.50%,and the induction rate of adventitious buds was 66.7%;3.The genetic transformation test of spinach mediated by Agrobacterium tumefaciens GV3101 showed that the callus induction rate of Sp75 leaf and petiole explants infected with GV3101 containing different recombinant constructs was only 1.0%,which is the induction rate of p BI121-GUS.Result;Adventitious buds were not induced in the experiment of direct infestation of callus;4.The infection experiments of LBA9402 with different recombinant constructs showed that the hairy root induction rate of Sp75 explants was 2.1% higher than that of Sp73,and the callus induction rate of Sp75 hairy roots was basically the same as that of Sp73;Induction efficiency and callus induction efficiency,p BI121-GUSSo Hsf B2 has the highest average induction rate of Sp75 hairy roots,p BI121-GUSSo Hsf A1 e has the highest induction rate of Sp73,p BI121-GUS-So Hsf A1 d has the highest induction rate of Sp75 callus The induction rate of Sp73 was the highest in GUS empty load;the induction rate of hairy roots of explants from different sources was significantly different.Under the overexpression of So Hsf A1 d,the induction rate of Sp75 explants was 4.3%-14.0%,Among them,petioles had the highest induction rate of 14.0%,Sp75 explants had the highest induction rate of 3.9% to 20.0%,and sedges had the highest induction rate of 20.0%;5.Bioinformatics analysis of SoHsfA1 d showed that SoHsfA1 d is homologous to Arabidopsis At Hsf A1 d,and compared with other species,there are certain differences in amino acid sequence,and motif analysis suggests that this difference is caused by the deletion or replacement of Motif 12 and Motif 19 motifs caused;6.Yeast two-hybrid system screened 8 candidate proteins and 3 candidate homologous proteins of reported interacting proteins with conserved domains directly or indirectly related to high-temperature stress.Still,they were found in heat-resistant material Sp75 and heat-sensitive material Sp73 There are different regulation modes of hightemperature response,indicating that So Hsf A1 d mediates different regulation modes in the two materials. |
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