| Maize is one of important industrial crop but stresess badly influence its growt h and development and restrict the yield and quality of maize.The establishment of arbsular mycorrhizal(AM)fungi and maize symbiot could not only enhance the a bility of arbsorbing mineral element and water in soil but also improve the stress t olerance and resistant.We have found that AM fungi inculation could promote the g rowth of the lateral roots of mazie.Therefore,we predicted that AM fungi inculation maybechange the redistribution of auxin of roots to induce the increasing the num ber of the lateral roots.It need to analyze the specific regulation mechanism further.The thesis,based on the symbiosis of AM fungi and maize,analysis the results of tr anscriptome to explore the key regulation network and then found few key membra ne protein genes involving the regulation of auxin redistribution according to the re sults of the bioinformatic the maize PIN genes family and AM fungi induced expre ssion pattern analysis.We verified the specific function choosed PIN genes in the pr ocess of the regulation of auxin redistribution and the growth of lateral roots by ha iry roots systerm of Lotus corniculatus in vitro and stable transformation of Oryza sativa.The main results are as follows:1.The lateral roots of mazie co-cultivated AM fungi for 30 days were sampled for transcriptome sequence in contrast to untreated root.Above 71% of clean reads can be mapped to mazie reference genome.23,997 genes were up-regulated during t he co-cultural processes compared to control without AM fungi inculation,while 3721 genes were down-regulated.GO term enrichment analyses showed that the DEG w as mainly related to ATP binding,DNA binding,metal binding etc.at molecular functi on level.At cellular component level,the DEG was mainly associated with menbrane.At biological process level,the DEG was primarily related to metabolic process,regul ation of transcription,DNA-templated and oxidation-reduction process,etc.KEGG path way enrichment analyses indicate that the DEG was mainly related to metabolism,pl ant-pathogen interaction and plant hormone signal transduction pathways.2.We filtrated the maize whole genome and obtained 14 maize homology PIN genes according to the characteristic of Mem_trans domain of PIN gene family and they were divided three subfamily according to the evolutionary relationship and th e sequence characteristics.The analysis of expression heat map showed that ZmPIN5 b and ZmPIN10 b do not express in all tissues but ZmPINY was opposite.ZmPIN1a-1d,ZmPIN8,ZmPIN10 a,ZmPINY genes expressed in primary roots of maize and ZmPI N1 a,ZmPIN1b was high expression especially.3.We analyze the induced expression pattern of auxin transporter PIN protein after AM fungi inculation by reverse transcription quantiative Polymerase Chain Rea ction(RT-qPCR).We found that the expression level of ZmPIN5 c,ZmPIN10 and Zm PINX genes were up-regulated differently after AM fungi and maize coculturation 30 days but ZmPIN5 c and ZmPINX genes were always up-regulated during the symb iosis and ZmPIN10 b gene only showed high expression level at early stage and unc hanged later.ZmPIN1 a,ZmPIN1c and ZmPIN9 genes were not induced by AMF.The expression level of ZmPIN1 b,ZmPIN1d,ZmPIN2 and ZmPINY genes was down-regula ted by AMF induced at the beginning but showed up-regulated later.4.We choosed ZmPIN1 a,ZmPIN5c,ZmPINY three PIN family genes accoding to the results of induced expression pattern to build the overexpression(35s)and indu cible expression(promoter of target genes)vectors.Then we connected the three PI N genes to plant expression vectors by PCR and enzyme digestion analysis and tra nsform into the Lotus corniculatus by agrobacterium rhizogenes-mediated method an d detected the positive hairy roots of transgenic Lotus corniculatus by PCR.Finally,we explored the role of key PIN genes in regulating the auxin distribution and the growth of the lateral roots after AM fungi inculation by agrobacterium tumefaciensmediated transgenic rice. |
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