| Lotus(Nelumbo nucifera Gaertn.)is an aquatic vegetable with the largest cultivated area in China.As one of the earliest domesticated crops,lotus has edible and ornamental value.Its seed and rhizome are main edible parts,and flower is the main ornamental part.The three cultivated groups were developed by artificial selection and natural selection.The comparative genomics study on lotus is helpful to understand the genetic differentiation and selection of the three cultivated groups.In the process of constant domestication,cultivated lotus showed some important selection characteristics:high yield,good taste,strong adaptability.The content of starch was the highest in edible parts of lotus.At present,there are many researches on the biological characteristics of starch in rhizome and seed of lotus,but few researches on the molecular mechanism of starch synthesis.Lotus seeds showed high protein content is parallel to dicotyledon seeds,and high starch content which is similar with monocotyledon plants.In addition,lotus seed showed high amylose content,is a good model species for studying high-amylose crops.In this study,several species of lotus were re-sequenced to obtain important information about the process of lotus domestication.Transcriptome analysis of developing lotus seeds to obtain some important data of material accumulation in the developing process.The cloning and functional verification of related genes of starch synthesis in lotus can provide guidance for molecular assisted breeding and quality improvement.1 A total of 69 lotus accessions were selected,including 45 cultivated accessions from three cultivated groups(21 seed lotus,13 rhizome lotus,11 flower lotus),22 accessions from wild sacred lotus,and 2 accessions from wild American lotus.With Illumina technology,the genomes of these lotus accessions were re-sequenced to>13×raw data coverage.On the basis of these genomic data,25 million single-nucleotide polymorphisms(SNPs)were identified in lotus.Population analysis showed that the rhizome and seed lotus were monophyletic and genetically homogeneous,whereas the flower lotus was biphyletic and genetically heterogeneous.Using population SNP data,we identified 1,214 selected regions in seed lotus,95 in rhizome lotus,and 37 in flower lotus.Some of the genes in these regions contributed to the essential domestication traits of lotus.2 Illumina HiSeq high-throughput sequencing was used to analyze transcriptome sequencing of four stages developing lotus seeds.Based on the gene/transcript expression changes analysis of various stages,we found genes with highly expressed and differentially expressed related to lotus seed morphological development.It laid a foundation for revealing the molecular regulation mechanism of lotus seed development.Among them,the highly expressed genes were mainly related to anti-stress related proteins,glycoanabolic proteins and storage proteins at each stage.The DEGs were mainly involved in biological processes such as glucose metabolism,signal transduction and starch synthesis.In addition,17 DEGs related to the starch synthesis pathway were analyzed which combined with morphological observation and histological section observation of various stages lotus seeds.It was found that the NnSSS genes which involved in amylopectin synthesis showed low expressed level low during the material accumulation period.Therefore,it was speculated that SSS was the rate-limiting enzyme of amylopectin synthesis,and low expression level during the material accumulation period was the main reason lead to decrease of amylopectin synthesis.3 Two SBE family genes,NnSBEI and NnSBEⅢ,were identified in lotus.Phylogenetic analysis sorted NnSBEI into SBE family B and NnSBEⅢinto SBE family A.UPGMA phylogenetic tree divided 45 individuals of lotus into three classes.The homozygous haplotype(A G G A G)of NnSBEⅢwas observed in seed lotus.During the seed embryo development stage,NnSBEⅢreached the peak in the middle of the development stage,while NnSBEI increased in the mid-late developmental stage.The different affinity activity of the two isozymes binding amylopectin and amylose assay indicated NnSBEI has higher activity and wider affinity.4 This study reports the cloning,expression and characterization of three different isoamylase genes,NnISA1,NnISA2,NnISA3 and one pullulanase gene,NnPUL from lotus.Sequence analysis shows that the similarity between the three ISAs is not high,but they have same structural functional domain which showed high conservative.The similarity of NnPUL compared with other species is higher,NnPUL has four function domain structure,and five conservative sequence,which indicated that it belongs to the typeⅠpullulan enzyme.The expression analysis of NnISA genes in different parts showed that NnISA1 and NnISA2 had the same expression pattern,but the same expression pattern was broken during the development of seeds,and the expression levels of NnISA1 and NnISA3 were higher than NnISA2.The enzymatic properties of NnPUL recombinant protein showed that the optimal temperature is 50℃,and the optimal PH is 7.2.The NnPUL protein retains pullulanase catalytic activities with K_m values of 2.02mg/ml for pullulan. |
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