Cloning And Expression Analysis Of Asparagine Synthetase Gene Of Cunninghamia Lanceolata(Lamb.)Hook.

Asparagine is an important amino acid during the the process of plant nitrogen metabolism,and is the main amino acid when nitrogen assimilated into amino acid.Compared with other amino acids,asparagine has a higher nitrogen and carbon ratio,stability and solubility,so it can serve as a vector to transport and store nitrogen in plants.It is a hoarder of ammonia and also a provider of ammonia,it can be an indicator to measure the nitrogen nutrition of many plants,and also can measure the protein content of many plants.Asparagine synthetase based on ammonia or glutamine and aspartic acid,catalyze and synthesis asparagine,it is encoded by a small gene family,because of the instability of asparagine synthetase,and there areasparaginase and inhibiting factor in the extracting solution,in order to know moreabout the function and character of the synthetase in physiology,people can use the molecular biological technique to clone the asparagine synthetase gene and study.There were no study reports about cloning the asparagine synthetase gene of Chinese fir so far,the main study of this article was to clone the asparagine synthetase gene of Chinese for seedlings,did the biology information analysis of the gene,test the expression quantity of the gene and the content of asparagine under different growth condition in Chinese fir seedlings.By exploring the expression pattern and function of the gene,researching the gene’s mechanism of action,the study’s goal was to provide the theoretical foundation to improve the nutrient use efficiency of Chinese fir and make it fast-growing,high yield and high quality.The main research results were as follows:1.Cloning of asparagine synthetase gene in Chinese fir seedlingsDownload some asparagine synthetase gene homologous sequences of some other plants from NCBI,design the degenerate primer of the conserved sequence of the gene,based on the Chinese fir seedling tissue,did the RNA extract,reversed transcription,and used the cDNA first-strand as template,did the PCR amplification,took back the Objective band and did the TA clone,then got the sequencing result.It has a length of 323bp,after blast,consider it as the conserved sequence of asparagine synthetase of Chinese fir.Used the RACE technique to do the full length amplification,and got the full length of cDNA by matching the sequences.The sequence’s length was 1609bp,there were a 1332bp length of ORF,a 127bp length of 5’UTR,a 150bp length of 3’UTR and a23bp length ofPoly(A),the ORF encoded 443 amino acid,the initiation codon was ATG and the termination codon was TAA.After blast,the gene sequence has a high homology with Picea sitchensis,pinus sylvestris,Glycine max and so on,so considered the gene as asparagine synthetase gene of Chinese fir.Degined the primer of the ORF,and obtained two ORF sequences of equal length,after translated into amino acid,we found that only the locus of 212,400,401,404,435 encoded different amino acid.In this study we called the two sequences ASN1 and ASN2.2.Bioinfortnatics analyses of asparagine synthetase gene of Chinese fir seedlingsThe amino acid sequences encoded by ASN1 and ASN2 were predicted a nd analyzed by bioinformatics software.The Result showed that:the molecular formula of the ASN1 and ASN2 vere C2223H3414N60O639S18 and C2221H3408N604 O638S18,they were all water loving,taking negative charge,instability and acidi ty proteins,didn’t contain signal peptide,were secretory proteins.They were tr ansmembrane proteins and may locate in the peroxysome.Serine,threonine and tyrosine may occur phosphorylation,and the phosphorylation probability and 1 ocus of serine were higher and more than others,so speculated that serine pho sphorylation site may regulate the comformational change of the proteins.The secondary structure of the proteins were random coil and α-helix,the tertiary s tructure were wreathed by the random coil and α-helix.There were no spiral c urls strcture of the proteins.The main function of the two proteins were synth esis of amino acids and meanwhile involved in transcription,translation and int ermediary metabolism.By building the evolutionary tree,we found it have a h igh homology between the two proteins and Picea sitchensis,Pinus sylvestris,Arabidopsis thaliana,Soybean,Astragalus sinicus,Cicer arietinum,Lotus cornic ulatus and Medicago.It had a nearer relationship with Picea sitchensis and Pin us sylvestris,and a farther relationship with Arabidopsis thaliana and Astragalu s sinicus.Based on the results of the bioinformatics analysis,ASN1 and ASN2 prot eins had the same physicochemical property,location,function,and structure,d o we consider the two sequences as the same gene.3.Analyses of quantitative expression of asparagine synthetase gene of Chinese fir seedlings under different growth conditionsBased on the full length of cDNA of asparagine synthetase gene,disigned the specific primers to do the fluorogenic quantitative PCR.The study used β-actin as reference gene,did the real-time fluorescent quantitative PCR by 2-△△C T analyzing the expression of the asparagine synthetase gene of Chinese fir se edlings under different growth conditions.The results were as follows:① The expression of asparagine synthetase gene were gradually increased during the f our stages that 24h after presoaking,budding,raddicle extension and grown int o the whole seedling.② Under 6h drought stress,there was no big change ofthe expression of asparagine synthetase gene,after 12h stress,the expression s ubstantial increased,and decreased after 24h,but still higher than the control.③ In the initial stage of aluminum stress,the expression of asparagine synthet ase gene significantly increased,then decreased after 6h and keeped stable,but still higher than the control.④ The expression of asparagine synthetase gene were higher than the control after 12h light stress and dark stress,and the exp ression under light stress was much higher than that under dark stress,after 24 h,the expression under two stress were all decreased,and light stress was mor e significant than dark stress.4.Analyses of the content of asparagine of Chinese fir seedlings under different growth conditionsIn order to analyze the relationship between the expression of asparagine s ynthetase gene and the content of asparagine of Chinese fir seedlings under dif ferent growth conditions,we test the content of asparagine as well as expressio n of asparagine synthetase gene,the results were as follows:① The content o f asparagine were gradually increased during the four stages that 24h after pres oaking,budding,raddicle extension and grown into the whole seedling.② The content of asparagine was increased significantly under 6h to 12h drought stre ss,decreased after 24h,but still higher than the control.③ In the initial stage of aluminum stress,the content of asparagine were gradually increased,then d ecreased after 12h,increased again after 24h,then decreased after 48h,but still higher than the control.④The content of asparagine were higher than the co ntrol after 12h light stress and dark stress,and the content were equal between light stress and dark stress.The content didn’t change when stress 24h,then decreased after 48h,but still higher than the control.Combining the change about the expression of asparagine synthetase gene and the content of asparagine,we predicted that the asparagine synthetase gene cloning in this study may involved in the regulation of stress condition,and t here may be more than one asparagine synthetase genes together involved in t he synthesis of asparagine in Chinese fir.