Identification And Analysis Of Mutants For OsASN1 Which Encoding Asparagine Synthetase In Rice

Rice is one of the most important crops in the world.It provides food for more than half of the world's population and accounts for 1/3 of the global cereal crop area.Nitrogen(N)is one of the major factors limiting plant development,and was considered one of the major factor restricting the yeild.Amino acid is the main form of nitrogen transport in plants.Nitrogen is absorbed by plants and assimilated into amino acid,which could be transported better and meets the demand for nitrogen in different tissues and organs.Asparagine is one of the main forms of nitrogen long-distance transportation in plants.Asparagine(Asn)is synthesized from glutamine(Gln),this transformation process is catalyzed by asparagine synthetase and ATP is necessary.Asparagine Synthetasel(ASN1)is responsible for the biosynthesis of asparagine following the supply of ammonium to rice roots.Although the expression and partical of the function of OsASN1 were reported,the affects of OsASN1 on pant development and the mechanism was not yet reported.In this thesis,OsASN1,which encodes asparagine synthetase in rice was studied.One T-DNA insertion in OsASN1 was screened and qRT-PCR,southern blot and backcross was done to confirm the T-DNA insertion line was knockout mutant.In order to explore the effects of OsASNl gene on rice growth and development,root phenotype,tiller bud growth,seed germination,nitrogen contents of plants,amino acid content in xylem sap and agronomic characters in field have been analyzed in OsASN1 mutant and it's wild type(WT).The main results were as follows:1.OsASN1 gene encodes a protein containing two conserved domains composed of 604 amino acids.OsASN1 may be localized on cytoplasm and nucleus.Bioinformatic and qRT-PCR analysis showed that the expression of OsASNl mainly on roots and is very low in shoots.The expression of OsASN1 is relatively high in the leaf blade,but it is very low in the leaves and root-shoot junction.Both nitrate and ammonium could induce the expression of OsASNl when supply them to rice roots,but the effect of the expression of OsASNl induced by ammonium was significantly higher than nitrate.2.In order to analyze the function of OsASN1,T-DNA insertion line was indentified by qRT-PCR,southern and backcross in OsASNl mutants.And OsASN1-CRISPR mutant also been constructed.3.The OsASNl mutants in comparison to WT showed great difference in phenotype:The seminal root of mutant was shorter than wild type,the lateral root density increased,the adventitious root number and total adventitious root length were significantly reduced,but there was no significant effect on the lateral root number,the tiller number of the OsASN1 mutants compared with WT showed significant reduction,but the formation of OsASNl mutants tiller bud were unaffected and the elongation of tiller bud were severely depressed;the germination rate of OsASNl mutant seeds was not affected,however,with the germination of seeds,the length of shoot and root were significantly shorter than that of the wild type;the destruction of OsASN1 gene resulted in a significant decrease in dry weight,and a significant decrease in total nitrogen content in shoot and root,but no significant difference in nitrogen concentration;at the same time,the content of asparagine in OsASNl mutants xylem sap decreased significantly,which indicated that OsASNl was responsible for the synthesis of asparagine in rice;the destruction of OsASN1 gene also depressed the reutilization of nitrogen and the transportation of nitrogen to spikelet,which resulted in a significant decrease in the number of grains per panicle and the number of two branches in rice.In conclusion,OsASN1 which encoding asparagine synthetase in rice plays an important role in the growth and development of rice.The destruction of OsASNl gene would depress the normal growth and development of rice,and eventually lead to the reduction of rice yield.