| Potato(Solanum tuberosum),an annual plant of the Solanum family,originated in South America,is one of the most widely planted high-carbohydrate crops in the world.Because of rich anthocyanin content in tubers,higher nutritional and health value than ordinary potato,advantages of deep processing and planting efficiency,purple potato has become one of the hot crops studied by the researchers.At present,it has started large-scale planting in Guangdong,Zhejiang,Shandong,Jiangsu,Sichuan,Chongqing,Yunnan,Gansu and other places in China.In this study,purple potato"Huasong 66"was used as the test material.Four random zone tests with different nitrogen application levels were set,including N0(CK):0kg/hm~2,N1:90kg/hm~2,N2:225kg/hm~2and N3:360kg/hm~2.The content of total anthocyanin in each treatment was determined and the difference of total anthocyanin in different nitrogen application levels was analyzed.In addition,the application of Illumina Hi Seq?2000 platform for four different nitrogen application levels of tuber transcriptome sequencing,combined with the related physiological and biochemical indexes and the verification results of real-time fluorescent quantitative PCR,select candidate genes associated with anthocyanin synthesis pathway.We also discussed the mechanism of tuber formation and anthocyanin accumulation of“huasong 66”in purple potato at different nitrogen application levels during tuber forming period.The results provide theoretical and practical basis for the efficient cultivation of purple potato in the future.The main results are as follows:(1)Agronomic characters and related physiological and biochemical characteristics at 4nitrogen levels.The results showed that the plant height was N1>N2>N3>N0(CK)as the nitrogen concentration increased.During the whole growth period,the relative content of chlorophyll generally decreased and increased with the increase of nitrogen concentration.Except for N0,anthocyanin content,flavonoid content and sucrose synthase activity all increased with the increase of nitrogen concentration.In different nitrogen treatments,they were as follows:N1>N2>N3.The anthocyanin content treated with N1 was 31.91mg/100g.The flavonoids content was 134.63mg/g.Sucrose synthase activity was 669.6 g/min/g.The activity of phenylalanine ammonia-lyase decreased first and then increased with the increase of nitrogen concentration.In different nitrogen treatments,the activity of phenylalanine ammonia-lyase in N0 was the highest,which was 1.76U/g.The activity of N2 was the lowest,1.24U/g.(2)Yield and quality performance at 4 nitrogen levelsAnalysis of yield and quality(reducing sugar,crude protein,starch and vitamin C)of four purple potato tubers treated with different nitrogen levels showed that N1 had the best comprehensive quality,and its crude protein,vitamin C and starch contents were higher than the N0(1.66%,20.52%and 17.40%,respectively).And its reducing sugar content was lower(0.31g/100g).Secondly,the comprehensive quality of N0 treatment is better.N3 had the worst quality,the lowest starch content and vitamin C content,only 10.26mg/g,9.84mg/100g,and the highest reducing sugar content,0.40g/100g.The yield of N2 was the highest,which was19808.55kg/hm2,132.67%higher than that of the control group,and the yield of N3 was16984.50kg/hm2,99.49%higher than that of the control group.In addition,the commodity rates of N2 and N1 were higher,and the differences between them were not significant,92.80and 92.79%,respectively.The second is N3,with a commodity rate of 87.31%.N0 had the lowest commodity rate,only 69.34%.(3)Nitrogen uptake and utilization at 4 nitrogen levelsNitrogen content of plants at 4 nitrogen levels was determined.The results showed that the nitrogen content of the whole plant increased with the increase of nitrogen application,namely,N3>N2>N1>N0.In addition,The nitrogen efficiency of purple potato was significantly affected by different nitrogen levels.The partial productivity of nitrogen fertilizer and the agronomic nitrogen utilization rate gradually decreased with the increase of nitrogen application,while the physiological nitrogen utilization rate first decreased and then increased with the increase of nitrogen application,among which N2 had the lowest nitrogen utilization rate.The nitrogen harvest index first increased and then decreased with the increase of nitrogen application,and N1 was the highest,30.88%higher than that control group.In conclusion,N1 treatment had the highest nitrogen utilization rate.(4)Analysis of gene differential expression of tubers at the early stage of formation at 4nitrogen levelsA total of 2836 differentially expressed genes were obtained by using high-throughput sequencing technology and bioinformatics to analyze the gene expression in tuber of purple potato.The number of different genes among the four groups(N0 vs N1,N0 vs N2,N0 vs N3,N1vs N2,N1vs N3 and N2 vs N3)showed that 454 genes were up-regulated and 361 genes were down-regulated in N0 vs N1.In N0 vs N2,78 genes were up-regulated and 118 genes were down-regulated.112 genes were up-regulated and 66 genes were down-regulated in N0vs N3.In N1 vs N2,22 genes were up-regulated and 78 genes were down-regulated.The most differentially expressed genes were N1 vs N3,among which 1259 genes were up-regulated and 1023 genes were down-regulated.In N2 vs N3,135 genes were up-regulated and 66genes were down-regulated.Therefore,the mechanism of the positive regulation of tuber growth and development of purple potatoes with low nitrogen and high nitrogen is more complex than that with no nitrogen application and low nitrogen.(5)Functional annotation and enrichment analysis of differentially expressed genes of tuber at 4 nitrogen levelsAccording to the GO classification and enrichment results of the differentially expressed genes in the four nitrogen application levels,23 biological processes,15 cell components and15 molecular functions were involved.In biological process,differential genes are mainly involved in metabolic process,biological regulation,cell process,stress process and tissue or biogenesis of cell components.In the cell components,differential genes were mainly involved in cell and membrane formation.In molecular function,differential genes are mainly involved in binding and catalytic activities.KEEG pathway enrichment shows some metabolic activity involved in plant body,such as carbohydrate metabolism,energy metabolism,secondary metabolites biosynthesis and metabolism of sugar and amino acid metabolism,there are also involved in defense related ways,such as benzene propane biosynthetic pathway,GSH metabolism pathway,plant hormone signal transduction,and so on.In addition,the genes involved in translation,transcription and other genetic information processing were also significantly enriched.In summary,different N application levels mainly affect the nutrient transport and metabolism-related genes in purple potato tubers by regulating signal transduction such as enzyme catalysis and binding and carbon and nitrogen metabolism.(6)Screening of genes differentially expressed in anthocyanin biosynthesis pathway in tuberThrough the tuber formation of different N application levels of anthocyanin differentially expressed genes related to the annotation and analysis,identified nine differentially expressed in total Unigenes may participate in the anthocyanin synthesis pathway,including structure genetic DFR(1),AT(1)and CHS(1),UFGT(2)and F3H(3),and the transcription factor LAR of the b HLH gene family.The expression of these candidate genes at different n levels was consistent with that of the total anthocyanin content at the physiological level,that is,the effect of different n levels on anthocyanin formation in tuber showed that low n level was conducive to the rapid accumulation of anthocyanin,while high n level was opposite.(7)Verify transcriptome sequencing resultsAccording to the annotation of differentially expressed genes in RNA-Seq sequencing analysis results.Ten candidate genes with significantly different expressions were selected for q RT-PCR verification,and the results are consistent with that of transcriptome sequencing. |
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