| The tubers of yam(Dioscorea opposita Thunb.)are rich in nutrients and can be used not only for medicinal and food purposes,but also as industrial raw materials and feed.Yam is an important economic crop in our country and widely distributed in Africa,Asia,Oceania and South America.Starch is the most important nutrient in yam tubers,and the level of starch content directly affects the quality and yield of yam tubers.Therefore,the study of starch accumulation mechanism of yam tubers for genetic improvement and the yield quality of yam has important significance.In this study,we used Bekeqi and Dahechengyu yam as test materials and measured the tuber morphology at different development stages and the physiological of starch accumulation;At the same time we used the transcriptomes way by combining RNA-seq and Iso-seq to sequencing that two species at seven developmental stages,screened the differential expressed genes related to starch accumulation through bioinformatics analysis;Based on the results of RNA-Seq analysis to ensure the key periods of starch accumulation and Omics sequencing Label-free proteomics,screened the differential expressed proteins related to starch accumulation through bioinformatics analysis;Through combined transcriptomic and proteomic analysis,we screened for differentially expressed proteins(genes)associated with starch accumulation.We used RT-PCR technology to clone the ORF of the FRK2 gene and performed bioinformatics analysis on the ORF of the gene;We constructed an instantaneous expression vector and injected it into tobacco leaves to perform subcellular localization of FRK2;We used q RT-PCR technology to analyze the expression of fructose kinase FRK2 gene in yam tubers at different developmental stages,organs,and stress conditions.We also analyzed the correlation between the expression level of this gene and the starch and sucrose content,and enzyme activity related to starch accumulation in the tubers;We constructed a plant expression vector for the FRK2 gene and transformed tobacco leaves to identify its function.This provides a theoretical basis for clarifying the mechanism of starch accumulation of the sucrose and starch metabolism during the swelling period of yam tubers,as well as for improving the quality and yield of yam tubers.The following conclusions were drawn from this study.(1)The activity of high-starch variety Bekeqi yam which include tuber sucrose synthase,fructokinase,adenosine diphosphate glucose pyrophosphorylase,starch synthase and α-amylase were significant higher than the low starch variety Dahechangyv yam;The dry matter content of tuber,sucrose synthase,fructose kinase,adenosine diphosphate glucose pyrophosphorylase,starch synthase and α-amylase activitiy were played a positively in regulating starch accumulation,sucrose content and sucrose phosphatase activity were played a negative role in regulating starch accumulation;The size of starch granules increased significantly at the peak of tuber expansion.(2)The results of RNA-seq analysis showed that 10419 differential genes were screened in the two varieties,of which the high-starch variety Bekeqi has 5478 differential genes and the low-starch variety Dahechengyv has 6849 differential genes;5177 differential genes were screened in the same period in the two varieties.The biggest difference in morphology between B-90 vs B-180 and D-90 vs D-180 were screened for1556 genes related to starch accumulation(contains 107 transcription factors),these genes were related to the progress of functions and metabolic,binding cell process etc;The biggest difference in starch content of D-150 vs B-150 was screened for 3557 genes related to starch accumulation(contains 160 transcription factors),these genes were related to the function and combination of genes,catalytic activity,metabolic process,etc.(3)The results of Label-free analysis showed that a total of 4374 differential proteins were screened in the two varieties;The key period with starch accumulation between B-180 vs B-120 and D-180 vs D-120 were screened for 2944 differential proteins related to starch accumulation(contains 5 transcription factors),the functional categories of these differential proteins were related to metabolic process,catalytic process,organic metabolic process,etc;The biggest difference in the starch content of D-150 vs B-150 were screened for 3229 differential proteins related to starch accumulation(contains 10 transcription factors),while the high-starch variety had more differential proteins,the functional categories of these differential proteins were related to metabolic process,catalytic activity,monobiotic process,etc.(4)The results of the joint analysis showed that a total of 3149 differential proteins(genes)were screened in transcriptomics and proteomics,including 458(14.54%)differential proteins(genes)were associated with starch accumulation.The result through validated by q RT-PCR showed that the expression of RNA-seq data matched 85.19%.(5)According to the joint analysis of transcriptome and proteomics,FRK2 gene was screened and the ORF sequence of the gene c DNA was cloned.The c DNA of this gene has a total length of 1002 bp and encodes 333 amino acids;The theoretical molecular weight of its encoded protein is 35.83 k D,with an isoelectric point of 5.27,and there is no transmembrane structure and it belongs to the hydrophobic protein category;The similarity of the FRK2 amino acid sequence between this gene and the 9 reported plants is over 82%,indicating a close evolutionary relationship with the turmeric.The results of subcellular localization indicate that the protein is localized in the cytoplasm and cell membrane.(6)The expression pattern of FRK2 gene was analyzed.The expression level of FRK2 gene is significantly positively correlated with starch content,highly negatively correlated with sucrose content and sucrose phosphatase activity and related to sucrose synthase,fructose kinase,AGPase,starch synthase ɑ-amylase activity is highly significantly positively correlated.(7)When FRK2 gene was transferred into tobacco for functional analysis.The starch content,fructose kinase and starch synthase activity of FRK2 transgenic tobacco were significantly higher than those of wild type tobacco,transgenic tobacco ɑ-amylase activity was significantly higher than that of wild-type tobacco,and the fructose content in transgenic tobacco was significantly lower than that of wild-type tobacco;Transgenic tobacco plants with FRK2 gene and wild-type tobacco plants were treated under low temperature stress,the starch and soluble sugar content,superoxide dismutase,peroxidase and catalase activity of transgenic tobacco were significantly higher than those of wild type tobacco,and the MDA content of transgenic tobacco was significantly lower than that of wild type tobacco,FRK2 gene was significantly induced to be highly expressed compared to untreated plants in transgenic tobacco leaves;Under drought stress on transgenic tobacco and wild type tobacco plants,the starch and soluble sugar content,superoxide dismutase,peroxidase and catalase activity of transgenic tobacco were significantly higher than wild type tobacco,the MDA content and water loss rate of transgenic tobacco were significantly lower than wild type tobacco,and the FRK2 gene was significantly induced in transgenic tobacco leaves.Overexpression of the FRK2 gene improved the starch synthesis ability;The gene can be induced by low temperature and drought to increase the starch content of transgenic tobacco. |
