The Mechanism Of Arbuscular Mycorrhizal Fungi Regulating Nitrogen Absorbption Of Lycium Barbarum

The research explored the effects of nitrogen and AMF（Arbuscular mycorrhizal fungi）inoculation on growth status,photosynthesis,nitrogen absorption and assimilation of Lycium barbarum L..The full-length sequence of nitrogen transporter genes was amplified and the gene expression of L.barbarum under nitrogen and AMF treatments was analyzed.The LbNRT3.1 of L.barbarum was introduced into tobacco to obtain transgenic tobacco plants overexpressing LbNRT3.1.The main results are as follows:1.Effects of AMF on growth status and nitrogen metabolism of L.barbarumThe results showed that the colonization rate of AMF increase with the increase of nitrogen concentration（NH₄NO₃ 0 m M,1 m M,15 m M）and the colonization rates were 29.32%,43.93%and 62.81%.AMF could promote the growth of L.barbarum by improving nitrogen absorption,assimilation capacity and photosynthesis rate.2.Cloning,bioinformatics analysis and expression analysis of nitrogen transport genes of L.barbarumTwo ammonium transporter genes（LbAMT1.1,LbAMT1.2）and three nitrate transporter genes（LbNRT2.4,LbNRT3.1,LbNRT4.6）was amplified by rapid amplification of cDNA ends.The open reading frames（ORF）of LbAMT1.1 and LbAMT1.2 are 1479 and 1556 bp encoding 490 and 517 amino acids respectively.The molecular weight of the protein is 52.6and 55.6 k Da,and the isoelectric point is 7.19 and 7.64;The ORF of LbNRT2.4、LbNRT3.1and LbNRT4.6 are 1593,1740 and 1746 bp encoding 530,579 and 581 amino acids respectively.The molecular weight of the protein is 57.6,65.1 and 63.9 k Da,and the isoelectric point is 8.92,8.87 and 8.78.LbAMT1.1,LbAMT1.2 and LbNRT2.4 were localized in the plasma membrane,and LbNRT3.1 and LbNRT4.6 were localized in the vacuole.The results of quantitative real-time PCR showed that nitrogen application increase the expression of LbAMT1.1,LbAMT1.2 and LbNRT4.6,while reduce the expression of LbNRT2.4.AMF inoculation increased the expression of LbAMT1.1,LbNRT2.4 and LbNRT3.1,indicating that AMF can up-regulate the expression of nitrogen transport genes to promote the nitrogen absorption of L.barbarum.3.Construction of transgenic tobacco overexpression LbNRT3.1By analyzing the restriction sites of the plant overexpression vector PROKⅡand LbNRT3.1,the upstream primers with the XbaⅠrestriction site at the 5’end and the downstream primers with the SmaⅠrestriction site at the 3’end were designed to amplify the full length of LbNRT3.1.Then it was inserted into PROKⅡby double digestion and T4 DNA ligation.After PCR verification and double enzyme digestion,the recombinant plasmid was introduced into tobacco by Agrobacterium-mediated genetic transformation to obtain transgenic tobacco overexpressing LbNRT3.1 through co-cultivation and screening culture.It lays the foundation for in-depth study of L.barbarum nitrogen absorption mechanism.