Transcriptome Analysis And Related Gene Mining Of Low Temperature And Weak Light Induced By Cucurbita Pepo

Cucurbita pepo(Cucurbita pepo L.)is a cultivar of the genus Cucurbitaceae.It is delicious and nutritious,and so is favored by consumers.However,in the process of the early spring plant cultivation,low temperature and weak light are the two main environmental factors in the production of Cucurbita pepo,which limit the yield and market duration of Cucurbita pepo.How to regulate the adversity stress encountered in production,so that it meets the needs of producers and consumers,it is necessary to analyze the characteristics of low temperature and weak light and its regulation mechanism,especially the functional analysis of important metabolic activities that play a regulatory role.In this experiment,the three-leaves and one-hearted Cucurbita pepo seedlings were respectively treated by low-temperature,weak-light,low-temperature and weak-light stress.The photosynthesis related parameters of the Cucurbita pepo seedlings were determined,and the degree of damage of the photosystem of the leaves under different stress conditions was determined.The morphological and physiological indexes at each time point were determined,and the redox state and the degree of cell damage in the leaves were investigated.The seedling leaves were subjected to transcriptome sequencing;GO enrichment analysis,COG classification and KEGG pathway analysis were performed on the data results;some differentially expressed genes were verified by real-time fluorescent quantitative PCR.The results of the verification study are as follows:1.Low-temperature low-light stress treatment has different effects on plant height,stem diameter,leaf area,root growth,flowering period and root crown ratio of zucchini,among which low-light stress treatment is beneficial to increase plant height,leaf area and seedling whiting length.Under low temperature and low light conditions,stem diameter,plant height,leaf area,root volume growth,average root diameter growth,root length and root-crown ratio of seedling of zucchini were significantly inhibited.The earliest flowering period of zucchini under single low temperature condition.2.Low temperature,low light and low temperature and weak light stress all caused photosystem damage of the leaves of the Cucurbita pepo seedlings.The non-photochemical quenching coefficient NPQ and the maximum photochemical efficiency of PSII in the dark reaction decreased significantly.Among them,the single weak light treatment has the largest decline.SOD,CAT,and POD are all enzymatic defense systems,and the content of malondialdehyde(MDA)can reflect the strength of response to adverse conditions.The results of this study showed that under low temperature and low light stress,the activities of CAT and SOD decreased gradually,while the activities of POD and MDA increased.Therefore,SOD activity,CAT activity and POD activity in cotyledons,and MDA activity treated by low temperature and low light,can be used as an indicator of resistance to squash.3.234,583 Transcripts were assembled,and the Transcript sequences were assembled again to obtain 83,650 Unigenes.Further,the assembled 83,650 Unigenes were compared in public databases such as NR,Swiss-Prot,GO,COG,KOG,eggNOG4.5,KEGG,and Pfam.There are 33,490 Unigenes with functional annotations,accounting for 40.03%of Unigene’s total.Analysis of the transcriptome data by three kinds of stress treatment showed that there were 1322 genes in the weak light group differentially expressed;1050 differential gene expression in the low temperature group and 1111 differential gene expression in the low temperature weak light group.Among them,there were 155 genes up-regulated under three stresses,156 genes were down-regulated in all three stresses,and 311 genes were differentially expressed,but the expression patterns were different.4.a functional annotation as Peroxidase(POD)gene(i.e,c47620.graph_c0)was screened from the transcriptomic sequencing of Cucurbita pepo.It was found that the length of sequence was 1245 bp,including a 996bp open reading frame(ORF),which encoded a protein of 331 amino acids.The protein had a predicted molecular weight of 35.70 kDa and a hypothetical isoelectric point of 5.40.The similarity of the proteins in the homologous proteins of the common fruits and vegetables,such as cucumber,melon and balsam pear,is 84%,84%and 81%.revealing that it was highly conservative.The GenBank accession of the gene was MF988285,and it was designated CpPODl gene.The real-time quantitative PCR analysis of the leaves of the squash seedlings treated with different stresses and confirming the differential expression of the gene showed that he CpPODl gene on the squash with low temperature and weak light treatment was higher than that untreated.The expression of CpPOD1 gene in single weak light or low temperature treatment was higher than that in low temperature and low light.The overall trend of POD enzyme activity was consistent with the expression of CpPODl gene.Both CpPOD1 gene expression and POD enzyme activity decreased firstly to a certain extreme value,and this extreme value appeared on the fifth day,indicating that POD activity was correlated with the low temperature and weak light stress of Cucurbita pepo.