Cloning And Functional Study Of Upstream Promoter Of Iron Deficiency Stress Expression Gene MxSAMS2 From Malus Xiaojinensis

Iron(Fe),a mineral nutrient necessary for plant growth and development,usually causes insufficient chlorophyll synthesis,resulting in chlorosis of leaf tissue resulting in poor crop growth.In extreme cases,iron deficiency may lead to a complete reduction in crop yields.It has brought huge economic losses to agricultural production.S-adenosylmethionine synthetase(SAMS)is an important enzyme involved in ethylene and polyamine synthesis in plants.In the early stage of the laboratory,S-adenosylmethionine synthetase(MxSAMS2)was cloned from Malus xiaojinensis.The function of MxSAMS2 under iron deficiency condition was studied.The results showed that the expression of MxSAMS2 increased under iron deficiency stress.The subcellular localization showed that when MxSAMS2 was transferred from nucleus to cytoplasm.MxSAMS2 transformed into Arabidopsis thaliana,under iron deficiency stress,the plants of MxSAMS2 showed stronger iron resistance than wild type plants.Moreover,the chlorophyll,fresh weight and root length of plants transferred to MxSAMS2 were all higher than wild type.However,the pathway of MxSAMS2 to participate in iron deficiency stress was not clear.In this paper,the promoter of MxSAMS2 upstream of Malus xiaojinensis was cloned,and the functional components of MxSAMS2 upstream promoter were analyzed.The main results obtained are as follows:1.the promoter of MxSAMS2 upstream was amplified by PCR,and the promoter was cloned into the vector p Cambia 1391 Z.The recombinant plasmid was constructed,named p Cambia1391Z-MxSAMS2-Promoter.The sequencing results showed that the promoter length of MxSAMS2 upstream was 2654 bp,which was transiently expressed by plasmid mediated injection of tobacco by Agrobacterium tumefaciens.It was found that MxSAMS2 promoter could activate GUS gene expression.It is proved that the sequence has the function of promoter gene expression.2.the prediction of cis acting elements in the Plant CARE and PLACE databases showed that the promoters contained 107 cis acting element elements: 4 phytohormone responsive components,5 biostress responsive components,27 abiotic stress responsive components,and71 essential components of the promoter.3.in order to further verify the prediction results of MxSAMS2 promoter function,we transformed the promoter into Arabidopsis thaliana and obtained positive seedlings.Exogenous abscisic acid(ABA)of 0 ??,50??,100??and 150?? was applied to the Arabidopsis thaliana seedlings after transformation promoter.The results showed that with the increase of abscisic acid concentration,the ability of MxSAMS2 promoter to drive GUS expression decreased gradually.The Arabidopsis thaliana seedlings after transformation promoters were treated with gibberellin(GA)and salicylic acid(SA)of 0 ??,100 ??,200??,400 ?? respectively.The results showed that MxSAMS2 promoter did not respond to GA and SA.4.the MxSAMS2 promoter was deleted from the 5 'end: full length(-2654 bp),fragment1(-1630 bp),fragment 2(-1190 bp),fragment 3(-723 bp),fragment 4(-342 bp),each fragment was fused with the GUS gene respectively.The recombinant plasmid of the fusion gene was transiently expressed in tobacco by Agrobacterium tumefaciens,and it was found that the activity of the gene was detected except for fragment 4.In order to observe the stability of the above 5 fragments,we transformed the fragments into Arabidopsis thaliana by dipping flower method,screened the Arabidopsis positive seedlings,and harvested the seeds of T1 generation.The positive seedlings of T2 generation were seeded.The seedlings of 10 days were seeded stain.The results of the test were consistent with those of tobacco transient expression.Besides fragments 4(-342 bp),other fragments could drive the expression of GUS gene.In this paper,the MxSAMS2 upstream promoter of Malus xiaojinensis was cloned.Firstly,GUS activity of the MxSAMS2 promoter was detected,and its sequence elements were analyzed.In addition,three plant hormones were selected to treat transgenic Arabidopsis thaliana,and the response of plants to hormones was observed.Finally,the promoter was deleted to determine the core region where the promoter functions.The research results laid a foundation for clarifying the regulatory mechanism of MxSAMS2 gene in response to iron deficiency stress,and also provided experimental basis for further breeding stable and efficient iron deficiency resistant genes.