Cloning And Functional Analysis Of Jasmonate Biosynthetic Pathway Key Genes NtAOC And NtAOS In Nitraria Tangutorum

Nitraria tangutorum is a shrub belonging to the Nitraria genus in the family Zygophyllaceae,usually distributed in the desert areas of northwest in China,having strong ability to resist high temperature,drought,cold,salinization,wind.Previous studies have shown that jasmonic acid,as a new plant hormones,plays an active role in resisting adverse environment in plant.In the present study,two key genes(NtAOC and NtAOS)related to biological synthesis of jasmonic acid were isolated from N.tangutorum and gene expression profiles were analyzed.In order to obtain NtAOC-and NtAOS-overexpression Arabidopsis,eukaryotic expression vectors were constructed and the transgenic lines were obtained.The functions of NtAOC in transgenic Arabidopsis thaliana participating in salt stress responses was verified.This study would be significant to the research of salt tolerance mechanism of jasmonic acid in Nitraria tangutorum.The main results are as follow:1.The NtAOC and NtAOS genes were cloned according to the data of transcriptome of Nitraria tangutorum.Bioinformatic analysis indicateted that NtAOC contains a 774bp open reading frame,encoding a protein with 257 amino acids.The ORF length of NtAOS is 1566bp,encoding a protein with 521 amino acids.NtAOC is close to the AOC in Catharanthus roseus,Pisum sativum,and Medicago truncatula.NtAOS is close to the AOS in the Citrus sinensis,Morus notabilis and Camellia sinensis.2.Quantitative RT-PCR results revealed that NtAOC and NtAOS were expressed in root,stem,leaf,flower and fruit.The expression levels of nutritional organs growed in field environment were higher than that of cultured plants.The expression level of NtAOC was highest in flowers and the lowest in stem.The NtAOC expression levels in different tissues of plants growed in field environment were:flower>fruit>leaf>root>stem.However,the NtAOS expression level was highest in root and lowest in fruit.The expression levels of NtAOS were:root>stem>flower>leaf>leaf>fruit.The expression levels of NtAOC and NtAOS were significantly increased in plants treated with low temperature,PEG,NaCl,H202,ABA and MeJA and it showed that NtAOC and NtAOS could be strongly induced by these factors.3.Overexpressing NtAOC and NtAOS in Arabidopsis thaliana and obtaining T3 transgenic plants,the function of NtAOC were verificated.The results showed that the susceptibility of NtAOC transgenic plants to salt stress were increased and the degrees of susceptibility were also rised with the increased salt concentration.Under 150mM NaCl treatment,the fresh weight,the contents of chlorophyll,proline,ABA and antioxidant enzymes(POD and CAT)activtity in the transgenic lines were significantly lower than the wild type,and the content of malondialdehyde(MDA)was significantly higher than that of the wild type.4.Using qPCR technique,the expression level of genes related to jasmonic acid biosynthesis(AtAOC2,AtOPR3,AtJAZ1),antioxidant system(AtCAT1,AtSODl,AtAPXl),proline synthesis(AtP5CSl),ion transport(AtSOS1,AtNHXl)and transcriptional regulation factors to stress(AtABF4,AtDREB2A,AtMYB2)were analyzed.The results indicated that the expression levels of jasmonic acid biosynthesis genes in transgenic Arabidopsis thaliana were significantly up-regulated.However,the expression levels of various stress related genes and the JA synthesis inhibitory protein gene were all significantly down-regulated compare with the wild type.It illustrated that the overexpressing NtAOC in Arabidopsis induced the expression of JA biosynthesis key genes,at the same time,negatively regulated the expression of stress-related transcriptional factor and resistance-related genes under salt stresses,and the transgenic plants showed more sensitivity to salt stress.