The CDNA-AFLP Differential Gene Expression Of Broccoli Leaves Under Simulated Acid Rain

China has become the third largest area polluted by acid rain after Europe and North America at present,pollution covering Guangdong,Guangxi,Hunan,Sichuan and other places,an area of over 200 square kilometers.It is the world's rare that acid rain area is expanding fast with high precipitation rate in our country.Acid rain can damage the growth of vegetables,reducing vegetable production.Vegetables leaves were under acid rain erosion would lead to chlorophyll content decreased,photosynthesis blocked.Injuries serious would cause atrophy and abnormal leaf.In order to study the effects of acid rain on the transcriptional level,reveal the mechanism of acid rain hazard better,provide a theoretical basis for vegetable production and the prevention of acid rain pollution,this research explored the effects of simulated acid rain on growth characteristic's of Broccoli seedling,and the differential gene expression analysis by cDNA-AFLP technique.the main research results are as follows:1.The reactive oxygen metabolism under simulated acid rain.Membrane permeability,MDA content and antioxidant enzyme activity changes of broccoli leaves under simulated acid rain treatment of 0,3,6,9 days,were explored.The results showed that with the increase of the concentration of simulated acid rain,the plasma membrane permeability of broccoli strengthened,the MDA content,SOD and POD activity increased gradually,the CAT activity decreased after the first upward trend.2.The changes in photosynthetic characteristics under simulated acid rain.The biomass,photosynthetic rate and fluorescence characteristics of broccoli leaves under simulated acid rain treatment of 9 days were explored.The results showed that with the increase of the concentration of simulated acid rain,the biomass decreased gradually,the photosynthetic rate(Pn),maximal photochemical efficiency of PS ?(Fv/Fo)and potential activity PS?(Fv/Fo)increased first and then decreased,the actual photochemical quantum yield of PS ?(?PS ?),photochemical quenching coefficient(qP)and non-photochemical quenching coefficient(qN)did not change significantly.3.Transcriptional level changes under simulated acid rain.The differentially expressed genes fragments of broccoli leaf under simulated acid rain stress were separated with using 192 pairs of primer combinations by cDNA-AFLP technique.Eventually 148 TDF were obtained with 126 of them sequencing successful.The results showed that:117 TDF were high homology with known sequences(E-Value<10-10)after removal of vector sequences and Blastn comparison.The TDFs were divided initially into nine categories according to different functions:electron transport,photosynthesis,membrane and transport,energy and metabolism,host defense,cell wall hydrolysis,signal transduction,transcription and translation gene,and some other genes of unknown function.4.?-1,4-xylosyltransferase IRX9H,ERF2,NAC,GST of broccoli related to resistance were obtained by RACE cloning technology,and analyzed by qRT-PCR.The expression of four genes under acid rain stress increased significantly at first,and then declined due to longer duration of acid rain stress,reached its maximum at 3d.The full-length of ?-1,4-xylosyltransferase IRX9H gene cDNA was 1550 bp,and the open reading frame has 1155 bases,encoded 384 amino acids,GenBank accession number:KF715851.The full-length of ERF2 gene cDNA was 958 bp,and the open reading frame has 804 bases,encoded 267 amino acids,GenBank accession number:KF715852.The full-length of NAC gene cDNA was 1055 bp,and the open reading frame has 903 bases,encoded 300 amino acids,GenBank accession number:KF715853.The full-length of GST gene cDNA was 912 bp,and the open reading frame has 642 bases,encoded 213 amino acids,GenBank accession number:KF715854.