| Chinese fir were often subjected to abiotic stresses such as drought and aluminum toxicity during the growth process.These abiotic stresses tend to cause irreversible damage to the Chinese fir and even death.Therefore,the abiotic stresses have huge effects on the Chinese fir about the plantation productivity and sustainable development.Studies have shown that asparagine is an important amino acid in the process of plant nitrogen metabolism,the main component of free amino acids,compared with other amino acids it has a higher nitrogen and carbon ratio,and more stable compared with others,so it can be used as plant nitrogen transport and storage tools.The Asparagine synthetase is an enzyme protein that catalyzes the synthesis of asparagine and can respond to different external stress conditions.Therefore,in this study,we analyzed the activity and function of asparagine synthase gene in non-biological stress,and analyzed the activity of asparagine synthase gene under abiotic stress.The use of transgenic technology to improve the traits of Chinese fir under abiotic stress and improve the resistance of Chinese fir to abiotic stress,and to provide a basis for further revealing the relationship between asparagine synthase gene and resistance to Chinese fir,Amide synthase gene and then cultivate a strong resistance to transgenic fir varieties to lay the theoretical basis.1 The cloning and bioinformatics analysis Chinese fir asparagine synthetase geneASN3 gene which was one complete ORF coding 231 amino acids was cloned from Chinese fir.According to bioinformatics analysis,protein encoded by ASN3 gene was stable and closed to water,without the signal peptide,winded helix and transmembrane domain,and had a variety of phosphorylation sites,with 15 serine,7 threonine and 3 tyrosine.The secondary structures and three-dimensional structure of ASN3 gene were mainly composed of random coil and alpha helix.2 Changes of asparagine synthetase activity in Chinese fir under abiotic stressThe experiment were conducted to study the effects of salt,sugar and nitrogen of abiotic stress on the activity of asparagine synthetase.The results showed that:① After 24 day of process of salt stress,the ASNS activity of Chinese fir was higher with the increase of stress time,and the ASNS activity reached the highest after 40 day which the NaCl concentration were fixed at 3.2 g/L.②under the abiotic stress process of sugar,the ASNS activity showed a trend of ascending and descending with the increase of the process time.When the sugar concentration was 50g/L and 70g/L,the ASNS activity showed that the ASNS activity increased first and then decreased when the sugar concentration was 90g/The ASNS activity was the highest at 24 days and 20 g/L.③under the abiotic stress process of Non-N,3/2N and 4/2N,the ASNS activity showed a trend of ascending and descending with the increase of stress time.Under the stress of 1/2N,the activity of ASNS showed the trend of ascending and descending.Under 4/2N stress,the ASNS activity of Chinese fir was the highest after 16 days.3 Quantitative expression analysis of asparagine syntheses gene in Chinese fir under abiotic stressThe experiment was conducted to study the expression of asparagine syntheses gene in Chinese fir seedlings under abiotic stressof salt stress,sugar stress and nitrogen stress.The results showed that under salt stress,with the extension of the stress time,asparagine syntheses gene increased first and then decreased under 0.8 g/LNaCl.But when the concentration of 1.6 g/L,it exhibit a variation trend of up-down-up;When the concentration of NaCl was 3.2g/L,it increased first and then decreased,at 24 day,the expression of small changes.The expression of ASN3 gene were up-regulated under salt stresses with various concentrations,while the expression of ASN3 gene showed no significantly differences among different salts treatments as compared with control during later-course of experiment.The sugar in stress,with the extension of stress,50 g/L and 70 g/L sugar,asparagine syntheses gene expression was first increased and then decreased,both at 24 days reached the maximum;90 g/L sugar,overall increased first and then decreased,especially in 8-16 days and 32-40 days two in the process of small changes in expression.Sucrose treatments also induced ASN3 expression to varying degrees,while during later-course of experiment the expression of ASN3 gene was suppressed.Under nitrogen stress,nitrogen deficiency inhibited the expression of the gene in the different stress period,with the increase of nitrogen concentration,the gene expression induced by nitrogen,4/2 N and 3/2N treatment conditions,this gene is up-regulated by inducing the expression of 1/and 2N were obvious.Nitrogen treatment significantly inhibited the expression of the gene.The responses of ASN3 gene to treatments with various nitrogen concentrations were different,nitrogen deficiency or low nitrogen treatment can induce the expression of ASN3,while high nitrogen treatments significantly suppressed its expression.4 The change of the Asn contents in Chines firThe Asn contents in Chines fir under salt,sucrose,and nitrogen stresses were determined by using euzymelinked immunosorbent assay.The results showed that the Asn contents were increasing to varying degrees under salt,sucrose,and high nitrogen stresses,while the Asn contents were decreased under nitrogen deficiency and 1/2 N treatment.5 Screening of antibiotics and concentration in genetic transformation of Chinese fir.In this study,the stems and shoots of Chinese fir tissue culture seedlings were used as plant materials,and different antibiotics were added on the basis of the basic medium:kanamycin(Km),hygromycin(Hyg)and Cef.The aim was to study the effects of different kinds and concentrations of antibiotics on the growth of stem and shoot of Chinese fir,and to determine the suitable concentration of these antibiotics in the genetic transformation of Chinese fir.The results showed that the survival rate of shoots and stem segments was only(8.78± 0.99)%and(13.97 ±4.56)%when 40 mg/L hygromycin was cultured for 42 days.When the hygromycin concentration was increased to 60 mg/L and 80 mg/L,the survival rate of shoots and stem segments of Chinese fir continued to decrease until their growth was completely inhibited.Therefore,40～60 mg/L and 60～80 mg/L hygromycin can be used as the selection pressure of the first and second screening of transformed seedlings during the genetic transformation of Chinese fir.When the concentration of Kana was 100mg/L,the effect on the growth of Chinese fir buds and stem was small,which was not suitable as the resistance of Chinese fir genetic transformation.Cephalosporin concentration of 300mg/L can effectively inhibit the growth of Agrobacterium tumefaciens GV3101,and in this concentration of Chinese fir buds and stem cells proliferation effect is good,the new growth of Chinese fir seedlings were dark green,grow well,can be used as antibacterial concentration.6 Study on Genetic Transformation System of Chinese FirThe ASN1 gene of Chinese fir was tansformed into tobacco by using leaf disc method.The transgenic tobacco plants were identified by PCR using ASN-ORFF、ASN-ORFR as primers,a segment that is consistent with the target gene size was get by PCR amplification,which indicating transgenic tobacco plants with Chinese fir ASN1 gene were generated.The transgenic plants with ASN1 gene were further tested for its aluminum tolerance,and the results showed that the contents of hydrogen peroxide and malondialdehyde were significantly lower in leaves of transgenic plants as compared with wild type,consisting with these results the expression of ASN1 in leaves of transgenic plants were significantly higher than wild type,the results suggested that transgenic plants with ASN1 gene showed a better aluminum tolerance capacity.The orthogonal experiment was used to study the effects of the treatments of ASN,OD600,soaking time,co culture time and AS concentration on the genetic transformation of Chinese fir.In the process of stripping resistance screening found in coniferous stems more earlier and withered phenomenon is more obvious,this may be due to the removal of needles not increase receptor material surface wound,conducive to the transfer of the recombinant plasmid,but also increase the wound physiological state of receptor materials produced adverse effects,thus increasing the sensitivity to antibiotics,the resistance in the culture conditions to normal growth,and ultimately death.In the experiment,the ASN gene was not successfully integrated into the stem of Chinese fir. |
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