Cloning And Functional Analysis Of L-galactose-1,4-lactone Dehydrogenase Gene In Strawberry Fruits

Vitamin C(also known as ascorbic acid)is a water-soluble vitamin that not only has a positive effect on preventing scurvy,arteriosclerosis and hypertension in humans,but also plays an antioxidant and antioxidant role in the growth and development of plants.The vital role of stimulus.In addition,vitamin C is also one of the standards for measuring the intrinsic quality of strawberry fruits.The content of vitamin C directly affects the nutritional value of strawberry fruits.Therefore,identifying key genes for vitamin C synthesis is of great significance for improving strawberry quality and its production.L-galactose-1,4-lactone dehydrogenase(Gal LDH)is a key enzyme in the synthesis of vitamin C,but the study ofGalLDH gene in strawberry fruits is rarely reported,so the mechanism ofGalLDH gene in the accumulation of ascorbic acid is elucidated.Is essential for strawberry production.In this experiment,a gene of L-galactose-1,4-lactone dehydrogenase was cloned from the fruit of ’Benihoppe’ strawberry,named FaGalLDH,and the gene was analyzed by bioinformatics.Chlorophenol indophenol titration method was used to analyze strawberry fruit development and ascorbic acid content in different organs;quantitatively analyze the expression pattern of FaGalLDH gene in real time;construct a prokaryotic expression vector to purify FaGalLDH protein,analyze its enzyme activity,and optimize the protein Optimal enzyme activity conditions;Constructing an overexpression vector to genetically transform Arabidopsis thaliana,and studying the function of FaGalLDH gene in transgenic Arabidopsis thaliana;In addition,the promoter of the gene was cloned from young leaves of ’Benihoppe’,and the promoter sequence was analyzed The elements can be used in combination with yeast one-hybrid technology to screen transcription factors that may regulate FaGalLDH gene.The main results completed are as follows:(1)The full length FaGalLDH gene was cloned from’Benihoppe’.Its CDS region is1743 bp and encodes 580 amino acids.It is predicted that its amino acid sequence molecular weight is 65.9 k D.It has a flavin adenine dinucleotide binding domain.(FAD-binging)and ALO(D-arabinono-1,4-lactone oxidase)domains,evolutionary tree analysis found that the strawberry FaGalLDH gene has the closest relationship with Prickly pear.(2)The results of the determination of ascorbic acid showed that the vitamin C content increased gradually during the ripening of the fruit;the analysis of the spatiotemporal expression pattern found that vitamin C was the highest in the mature fruits,followed by leaves,roots,stems,flowers,Very little.The expression pattern of FaGalLDH gene was positively correlated with the content of vitamin C,and the expression level gradually increased as the fruit matured;the expression pattern in organ tissues was also consistent with the content of vitamin C.In addition,among the four strawberry varieties,the expression of FaGalLDH gene was significantly correlated with the level of ascorbic acid;Benihoppe’ strawberry fruit had the highest vitamin C content,followed by ’Akihime’,’Ssanta’,and ’Jingjiao Xiaobai’.(3)The activity of L-galactose-1,4-lactone dehydrogenase expressing FaGalLDH protein was analyzed.The FaGalLDH protein was expressed and purified in prokaryotic cells.Analysis showed that the optimal activity of the protein was about25 °C,and the optimal p H was 4.0.In addition,in different stages of strawberry development and in different organs such as roots,stems,leaves,flowers,and mature fruits The expression pattern of the enzyme activity is similar to that of vitamin C content and the expression pattern of FaGalLDH gene.These results indicate that the expression of FaGalLDH gene is closely related to the protein activity of FaGalLDH and the accumulation of ascorbic acid content.(4)Functional verification of overexpression of FaGalLDH gene in Arabidopsis thaliana: The ascorbic acid content of the two FaGalLDH transgenic Arabidopsis thaliana plants was increased by approximately 73 % and 30 %,respectively.The expression of FaGalLDH gene and the enzyme activity of FaGalLDH were higher than those of the control.Wild Arabidopsis thaliana,and the semi-quantitative results also showed that the transgenic Arabidopsis thaliana PCR product bands were brighter than wild Arabidopsis thaliana,indicating that the strawberry FaGalLDH gene was overexpressed in Arabidopsis thaliana,increasing the plant’s FaGalLDH protein activity and ascorbic acid content.(5)The results of Arabidopsis thaliana treated with different concentrations of sodium chloride showed that at 50 m M,100 m M,and 150 m M sodium chloride concentrations,the root length of overexpressing plants Line1 and Line12 was significantly longer than that of control plants.(6)Cloning the FaGalLDH gene promoter and Screen the library using Yeast one-hybrid system.As a result,26 PCR products with different band sizes were screened.After sequencing and NCBI database Blast,There are 12 possible candidate genes such as the main transcription factors,phosphorylating enzymes,modified protease.