Cloning And Stress Response Analysis Of An ABA Pathway Gene AhLOS5 In Arachis Hypogaea L.

Peanut(Arachis hypogaea L.)is one of the most important oil crop and economic crop in China.With its deep rooting,no obvious critical period of water requirement,highly water use efficiency and drought resistance,peanut usually be chosen as an ideal crop for developing dry farming.Drought is often accompanied by desertification and salinization,bring a variety of factors restricting agricultural production.Two-thirds of peanut are grown in dry land.Lacking irrigation conditions and barren soil makes drought and high salt damages very common,which had already become the major limitation that most harmful and widely-spread.In China,70% of peanuts grow under various degrees of drought and high salt,which causes more than 20% yield loss every year.The responses to abiotic stresses are accompanied by multiple gene regulation in the plant development.Abscisic acid is involved in many physiological responses to environmental stresses,including drought,cold and high salt.Currently,ABA has been recognized as an anti-stress inducing factor of plants,which is able of inducing resistance to adverse environments.It is acknowledged that the endogenic ABA is synthetize via indirect biosynthesis pathway,and the ZEP,NCED,AAO and MCSU are the key regulatory enzymes.In our research,we cloned the MCSU gene AhLOS5 in a cultivated peanut species Fenghua1,and studied the expression characteristics of it under different inducible conditions.Two different plant expression vectors containing AhLOS5 gene were constructed and then introduced into Samsun by Agrobacterium tumefaciens-mediated transformation.Many transgenic plants of different expression levels were obtained.We studied the function of T1 generation of the transgenic plants.The results are as follows:1.We performed a drought resistance identification between 4 peanut main cultivars,and the ranking is Huayu20> Fenghua1> Haihua1> Baisha1016.2.We cloned a AhLOS5 gene from Fenghua1 with the RT-PCR and RACE methods.The 2731bp-length gene has a 115bp-length 5?noncoding region and 165bp-length 3? noncoding region.The ORF is 2451 bp,coding a protein of 816 amino acids,MCSU.3.Using Northern blot analysis with specific probe according to the AhLOS5 cDNA sequence,the result revealed that the expression level is Huayu20> Fenghua1> Haihua1> Baisha1016.The expression level is entirely consensus to the drought resistance ranking,which proved its drought resistance in peanut.4.We studied expression character of AhLOS5 at mRNA level under different inducible treatments in Fenghua1 peanut with Northern blot.The result revealed that AhLOS5 is a constitutive gene,the expression level ranking is flower>mature leaf> young fruit> fruit needle> stem> root.The expression levels in leaves increased with the leaf age.The expression of AhLOS5 was also remarkable induced by drought and salt stress.5.We constructed two different expression vectors,that is the pBI-LOS5 vector containing sense AhLOS5 gene,and pBI-R-LOS5 vector containing antisense AhLOS5 gene.Transformed these vectors into tobacco by Agrobacterium tumefaciens infection.The results of kanamycin resistance selection transgenic seeds of T0 generation and the PCR assay of progeny T1 of the transgenic plants all proved that the heredity property of transgenic progenies is coincident with the 3:1 separation ratio.6.We analyzed the root length,the content of MDA,ASA,endogenic ABA,proline,the activity of SOD,POD,CAT and APX,the results show that,overexpression in transgenic plant results in enhanced drought resistance,with increased ABA content,osmolyte content,antioxidant enzyme activity and membrane integrity.7.We analyzed the expression level of three other resistance genes,DREB2 B,RD22 and P5 CS.The result shows that all three resistance genes are inducible by drought and high salt in sense transgenic plants or WT plants.Conversely,in the antisense transgenic plants,only the ABA-independent resistance gene DREB2 B is inducible by drought and high salt,the ABA-dependent resistance genes RD22,P5 CS are not inducible.These results revealed that AhLOS5 is involved in the ABA-dependent resistance pathway,the resistance of transgenic plants to drought and high salt is regulated by the stress-induced endogenic ABA.