Cloning And Genetic Transformation To Arabidopsis Of APS Reductase Gene (APR2) In Rapeseed

Rapeseed is one of the four largest oil crops planted and its oil is an important source of edible vegetable oil in China.Reasonably rapeseed production occupies an important position in the national economy.To enrich the knowledge of molecular mechanism of purple-leaf trait formation and gene pool,a stable purple-leaf rapeseed line XY-PL selected by our laboratory was used as the experimental material in this study.First,the morphological characteristics,main agronomic characters and environmental impact factors of the purple-leaf trait were investigated,in addition,the inheritance pattern of the trait was also explored.Second,a reported candidate gene Adenosine-5'-phosohosulfate reductase 2?APR2?was cloned by homologous cloning method.Subsequently sequence analysis and functional prediction of this gene were performed applying bioinformatics at nucleotide and amino acid levels.Finally,3homology APR2 genes separated from XY-PL and ZS11 were constructed into plant expression vector,then following the Agrobacterium mediated transformation to Arabidopsis by using floral dipping method.On the basis of kanamycin selection and PCR verification,the leaf color of T1-T2 transgenic lines were investigated comparatively under purple-leaf inducing conditions,other phenotypes of these transgenic lines culturing without sulfur and high sulfur conditions were also discussed.The main results are summarized as follows:1.The leaves of XY-PL seedlings exhibited obviously purple color,and the color prolonged throughout the life cycle.XY-PL adult plant was about 1.6 m tall,had 4yellow petals and a compact plant type.The basal leaf of XY-PL was lobed with serrated margin and medium thickness.The leaf upper surface and veins were dark purple while the lower surface was light purple or green throughout the life cycle.XY-PL plant had upright,slim,green stem covering with wax powder.Comparatively,XY-PL plant had fewer branches and lower yield per plant than that of the control ZS11.Under Brassica juncea genetic background,the purple-leaf was dominant to green-leaf,and the populations of F₂ and BC₁ segregated in the ratio 3:1 and 1:1?purple to green?respectively,which showed the purple-leaf trait was controlled by a dominant gene and inherited in a complete dominance pattern.Whereas under Brassica napus genetic background,an intermediate type of light purple plants appeared in the F₂ population and the segregated ratio didn't comply with 3:1?purple to green?,inherited in a incomplete dominance pattern.2.Microscopic observations revealed that purple cells of XY-PL were mainly distributed in the epidermis under the cuticle and the adjacent palisade tissue.Using ZS11 as the control,4 leaf pigments?anthocyanin,chlorophyll a,chlorophyll b and carotenoid?were extracted and detected.The results showed that the average anthocyanin content in XY-PL leaves was 0.869 mg/g,the highest was 1.815mg/g,much higher than 0.025 mg/g of control;The average chlorophyll content and carotenoid content were 1.19 mg/g and 0.39 mg/g,lower than 1.59 mg/g and 0.44 mg/g of control respectively.Further anthocyanin stability tests demonstrated that anthocyanin extracted from XY-PL was still stable in acidic solution?pH?5.0?and below 45?.Under the conditions 60%light intensity,11h/13h?L/D?photoperiod,13??day?/8??night?temperature,XY-PL seedlings could stably express purple leaf trait.3.Three APR2 homology sequences were identified from rapeseed,including APR2-22?1365 bp?from XY-PL,and APR2-15?1374 bp?and APR2-19?1371 bp?from ZS11.Bioinformatics analyses revealed that APR2-22 encoded 455 amino acid residues?AAs?with molecular weight 50426.25.APR2-22 had a 64 AAs typical chloroplast transit peptide?CTP?at the N end and was a chloroplast targeting protein with unstable,non-secreted and hydrophilic characteristics,While APR2-15 and APR2-19 had the same 67 AAs length CTP with 3 more AAs?SLK?at positions 51-53.Structural analyses showed that APR2-22 consisted of 36.87%?-Helix,5.74%?-turns,41.28%random coil and 12%extension chain,in which four conserved domains5'-adenylylsulfate reductase,5'-phosphosulfate sulfotransferase,adenylyl sulfate?PAPS?reductase and 5-adenylylsulfate?APS?reductase were discovered.BLASTp results demonstrated that all the three APR2 protein sequences had a high similarity to5-adenylylsulfate?APS?reductase of cabbage,radish,kale and Arabidopsis.4.Three over-expression vectors of APR2 genes mentioned above were constructed into pCAMBIA1300,and each was transformed to Arabidopsis thaliana?col-0?by using Agrobacterium-mediated floral-dipping method.Four?APR2-22?,three?APR2-15?and two?APR2-19?transgenic lines were identified with the help of PCR detection and kanamycin selection.T2 seedlings of APR2-22 transgenic lines showed no purple leaf phenotype under purple-leaf inducing conditions,neither did the APR2-15 and APR2-19transgenic lines,which implied that APR2-22 was not enough to determine the purple-leaf phenotype,some other effectors were also needed.The average anthocyanin content of APR2-22,APR2-15,APR2-19 transgenic plants and wild-type plants were0.0223 mg/g,0.023 mg/g and 0.0248 mg/g respectively,there was no significant difference among them.5.The phenotypes of APR2 transgenic and wild-type plants cultured in nutrient solution with different sulfur contents were investigated.The results showed that wild-type plants cultured in MS normal solution had earlier flowering time to those cultured in high-sulfur and no-sulfur solutions,and had a better plant height,branch number,leaf number and pod number comparatively,which suggested that high-sulfur and no-sulfur environments could repress the growth of plants.In addition,the phenotypic and physiological differences among APR2 transgenic lines were less than that of wild-type plants cultured in solutions supplemented with high-sulfur or no-sulfur,which demonstrated that over-expression of APR2 in plants could improve tolerance to sulfur in the environment.