Cloning And Functional Verification Of Asparagine Sythetase Genes Of Mulberry

Mulberry（Morus notabilis L.）is an economic perennial woody plant with a long history of cultivation.Mulberry leaves,which account for about 60%output of the above-ground part are the main product of mulberry trees.Mulberry leaves contain diversified and balanced nutrients,such as high crude protein content,carbohydrates,vitamins,alkaloids,etc,which make mulberry leaves the best provender to feed silkworms（Bombyx mori L.）.Nitrogen metabolism plays an important role in the life activities of mulberry.Asparagine Synthetase（AS）exists in plants and animals.It catalyzes the conversion of aspartic acid（Asp）to asparagine（Asn）with the amino from glutamine（Gln）or ammonium in nitrogen metabolism pathway.In many plants,Asn is the main recipient of the amino from Gln.Asn has high stability,carbon-nitrogen ratio and strong solubility.It is the main form of transportation,storage and redistribution of plant nitrogen between sources and sinks.It has relatively high levels of free amino acids in the phloem of many plants.In order to explore the function,role of AS in nitrogen metabolism in mulberry,we cloned the AS genes from Morus notabilis,analyzed its predicted structural characteristics,explored its expression under stress conditions or during different developmental stages or in different organs of mulberry.We concluded that AS plays an essencial role in the growth and development and adversity of mulberry.The Arabidopsis mutants were used to further characterize the function of plant AS in growth and development and stress adaptation.The results are as follows:1 Cloning and bioinformatics analysis of the MnASNs gene familyTwo ASN genes were identified in Morus Genome Database.Primers were designed based on those sequences,and two MnASNs genes were cloned using the c DNA reverse-transcripted from the winter bud of Morus notabilis as a template and were named MnASN1 and MnASN2,respectively.The full length of the CDS sequences was 1761,1758 bp encoding for proteins with 586 and 585amino acids,respectively,and the predicted protein molecular weight was 65.58 and 65.80 k Da,and the PI value is 5.97 and 6.07,respectively.Gene structure analysis showed that each of MnASN1 and MnASN2 contained an open reading frame,MnASN1 contained 13 exons and 12 introns,and MnASN2 contained 14 exons and 13 introns.MnASN1 contains 18 Asn residues and MnASN2contains 14 Asn residues.The amino acid sequence of AS was highly conserved among isozymes among different species,and the last 40 amino acids at the C-terminus are relative less conserved area.The promoter elements of MnASNs varied in number and types.Phylogenetic tree analysis showed that the ASN homologs of angiosperms were divided into two families:MnASN1 belongs to Class 1;MnASN2 belongs to Class 2.2 Subcellular localization of MnASNsDifferent programs were applied to predict the subcellular location of MnASN proteins,the results from Plant-m PLOC online software showed that the MnASN1 protein was located in the chloroplast and MnASN2 protein was located in the cytoplasm.While the online software Wo LF PSORT II Mnkes predictied that MnASN1 is located in the cytoskeleton,cytoplasm,chloroplast and vacuole membrane,and MnASN2 is located in the cytoplasm,chloroplast,nucleus and vacuole membrane.The results of transient expression of MnASNs in tobacco leaves showed that MnASN1protein was localized in the cytoplasm,and MnASN2 protein as well.However,a large amount of protein agglutination was also found at the cytoplasmic edge,which might give hints that MnASN2might participate in the secretion of extracellular functions.3 Expressional patterns of MnASNs in growth and environmental adaptation（1）MnASNs exhibit high tissue expression specificity.The expression of two genes was higher in phloem and roots.The overall expression level of MnASN2 is higher than that of MnASN1.（2）During the germination of mulberry seeds,the expression level of MnASN2 was significantly higher than that of MnASN1,and reached the max value 10 days after sowing.（3）The expression of MnASN1 decreased significantly after the addition of any of NH₄Cl,Asp or Gln,and was particularly sensitive to NH₄Cl;the expression of MnASN2 decreased significantly after the addition of NH₄Cl,but increased after the addition of Asp and Gln for 24 h.（4）Under drought stress,the expression of MnASN1 responds first and MnASN2 responds later.On the 5th day of drought,the expression of MnASN1 is significantly reduced,while the expression of MnASN2 is up-regulated by about 40 times compared with the control group.Before 72 hours of salt stress,the expression of MnASN1 was higher than MnAS2,and after 72 hours,the expression of MnASN1 decreased,and the expression of MnASN2 increased significantly after 72 hours.（5）The expression of MnASN1 and MnASN2 is also affected by the circadian rhythm.4 The relationship between mulberry crude protein accumulation and asparagine synthaseThe crude protein content of mulberry leaves decreased with the increase of leaf position.Detection of enzyme activity related to nitrogen metabolism showed that the enzyme activity of AS in leaves was the highest,and the enzyme activity from high to low was:AS>GS>NIR>NR.The enzyme activity of GS decreased with the increase of leaf position,which was similar tendency as the changes of crude protein content in leaves of different positions.The expression of MnASN1decreased with the increase of leaf position,which was also consistent with the changes of crude protein contents in leaves of different positions.The crude protein content of mulberry leaves was significantly different in different varieties,from high to low,the order was:guoshusang>hongguoyihao>5801>shishengsang>dashi>zhenzhubai.The order of AS enzyme activity from high to low was:guoshusang>honguoyihao>5801>zhenzhubai>shishengsang.The level of AS enzyme activity is not completely consistent with the changes of protein content in different varieties.There is no significant difference in the expression of MnASN2 between varieties,but the expression level of MnASN1 among different mulberry varieties is significantly different.The expression level of MnASN1 among different mulberry varieties is from high to low as follows:guoshusang>hongguoyihao>5801>zhenzhubai>shishengsang>dashi,and that is a similar trend of the total crude protein content among different varieties.Therefore,the protein content accumulation in leaves might have a certain relationship with the level of ASN enzyme activity and the expression level of MnASN1.The accumulation of mulberry crude protein is significantly different in different seasons.From mid-July to early December,the crude protein content in mulberry leaves decreases.From the end of September to early December,the crude protein in the phloem and xylem at the base significantly increased,that is,as the winter comes,the proteins synthesized in the mulberry leaves are redistributed and transferred to the xylem and phloem for storage.It was found that the AS enzyme activity in xylem and phloem was significantly higher than that in young leaves,buds and mature leaves.From mid-July to mid-November,the AS enzyme activity in the phloem and xylem of the guoshusang continued to increase,while the AS enzyme activity in the leaf continued to decrease.It is speculated that AS might be involved in the reuse of nitrogen in mulberry trees.The expression level of MnASN1 is basically consistent with the trend of crude protein accumulation.It is speculated that MnASN1 is constantly expressed in the phloem and xylem to produce AS for the reuse of nitrogen in the phloem and xylem of mulberry.5.Researches on the functional verification of ASN in ArabidopsisThe homozygous mutant of asn2-2 was identified by the three-primer method.Compared with the wild type,the expression level of At ASN2 in the leaves of the 45-day asn2-2 mutant was only 1/6of that of the wild type（WT）.The first and second leaf positions of the mutant plants were different from the wild type in leaf size and shape.The mutant plants were smaller and their leaves were yellower than WT.The chlorophyll content,nitrogen content and fresh weight of the mutant plants were significantly lower than those of WT,and the mutant plants showed an apparently early flowering.Under high salt and drought treatments,the asn2-2 mutant displayed a severe wilting than WT under stress.The MDA content of the asn2-2 mutant was higher than that of the wild type under stress condition but same under normal growth condition.The soluble sugar content,SOD activity,and proline content of the asn2-2 mutant were lower than that of the wild type.In conclusion,the deletion of the ASN2 gene reduced the resistance of Arabidopsis to salt and drought stress.Under drought stress,At ASN1 expression level was significantly up-regulated in WT and asn2-2,and more induced in asn2-2 than WT.Under mild and moderate salt stress（50,100 m M Na Cl）,the expression of At ASN1 and At ASN2 were significantly up-regulated in WT and mutants.With the increase of Na Cl concentrations,the expression levels were also up-regulated.The expression level of At ASN1in asn2-2 is highly up-regulated than that in WT.The research results are basically consistent with the function speculation of AS of mulberry.