| Chilling stress is an abiotic factor limiting the growth,development,distribution and yield of plants.During chilling stress,a number of the major biological processes,such as photosynthesis,protein synthesis,lipid metabolism and secondary metabolism are affected.The first component damaged by chilling stress is the chloroplast membrane.Chilling tolerance is related to the composition and structure of plant membrane lipids by the transition from a liquid-crystalline phase to a gel phase.Tolerance to chilling stress is closely connected with the unsaturated fatty acid content of plant membrane lipids.Maize?Zea mays L.?is an important food crop,forage crop and industrial raw material in China,playing an important role in agriculture and the national economy.The accumulated temperature of the whole growth period is more than 10?.The optimum growth temperature is between 25?and 28?.When the temperature is lower than 12?,the plants are vulnerable to be damaged by chilling stress.Low temperature can decrease maize production by affecting seed germination and seedling growth,especially at early spring.However,the mechanism of chilling tolerance in maize is still unknown.In this study,physiological,transcriptome and molecular mechanism of chilling tolerance in maize were analyzed.Moreover,the key genes involved in chilling tolerance in maize were screened.Results of this study provide more valuable information for understanding the complex regulatory networks underlying the mechanisms of chilling tolerance in maize.The main results are as follows:?1?Screening for chilling tolerant and sensitive inbred maize line.Effects of chilling treatment?4?for two weeks?on the phenotype of 102 maize inbred lines were observed.By observing the phenotype,we preliminarily screened four chilling tolerance maize inbred M54,W54,287M,24034 and four chilling sensitive inbred line 753F,24045,KWS9384M,B7.Effects of chilling stress on shoot,root fresh weight and dry weight,plant height and leaf area of the 8 inbred lines were determined.Growth parameters of these 8inbred lines under chilling stress were quantified by membership function method.Among them,chilling tolerant inbred line M54 and chilling sensitive inbred line 753F were selected for following experiments.?2?Chilling resistant ability between M54 and 753F were analyzed.Effect of chilling stress on Fo,Fv/Fm for 0h,4h,8h,12h and 24h,O2·-and H2O2 content,flavonoid and total phenols content and fatty acid content were determined.Results showed that PS?of M54 was less affected than that of 753F by chilling stress.Antioxidant activity and secondary metabolite accumulation were higher in M54.The saturated fatty acid content decreased and the unsaturated fatty acid content increased,which increased the chilling tolerance of maize.?3?The molecular mechanism of chilling resistant in maize was preliminary determined by transcriptome.Total RNA was isolated from maize leaves of M54 and 753F treated with 4?for 4h and24h,which was used for transcriptome analysis.1665 and 3970 genes showed differential expression levels in M54 at chilling stress for 4h and 24h,respectively.While in 753 F,there were 547 and 1766 genes showed differential expressed at chilling stress for 4h and 24h,respectively.The gene annotation and classifications were compared with databases of NR,Swiss-Prot,GO,COG,KOG,Pfam and KEGG.We determined that many differentially expressed genes were involved in pathways related to phenylpropanoid biosynthesis,phenylalanine metabolism,starch and sucrose metabolism,and carbon metabolism.This showed that secondary metabolism and carbohydrate metabolism played important role in the chilling tolerance.Accordding to our RNA-seq data,DEGs related to carbon fixation in photosynthetic organisms were all up-regulated in both genotypes.There were 4 and 8 DEGs up-regulated in M54 after treated for 4h and 24h under chilling stress.While in 753F,only 1 gene related to ribose-5-phosphate isomerase was up-regulated at chilling stress for 24h.In the lipid mechanism pathway,there were 4 and 1 DEGs related to unsaturated fatty acids biosynthesis up-regulated in M54 and 753F,respectively,under chiling stress for 4h.Benzoxazinoid biosynthesis related DEGs were only found in M54.Many genes related to phenylpropanoid biosynthesis showed differential expression under chilling stress.Most of these DEGs were up-regulated,especially in M54.?4?Functional analysis of genes related to chilling tolerance in secondary metabolism pathway.According to our RNA-seq data,we selected two MYB-IF35 and MYB31 genes for following experiments.These genes differentially expressed in M54 under chilling stress.The bioinformatic analysis of MYB-IF35 and MYB31 using TMHMM,SMART,ExPASy and other online software groups predicted its protein structure and physical and chemical properties,which laid the foundation for further study of its function.The full length of MYB-IF35 and MYB31 were cloned from maize cDNA.The subcellular localization result showed that maize MYB-IF35 and MYB31 localized in nucleus.The full-length of MYB-IF35 and MYB31 was subcloned into the expression vector to form sense construct of pMWB110-MYB31 and pMWB110-IF35.The construct was introduced into Agrobacterium tumefaciens strain EH105.And the vector is transforming into maize by Life Science and Technology Center,China Seed Group Co.,Ltd.We also formed sense construct of pROK?-MYB31 and pROK?-IF35.The constructs were first introduced into Agrobacterium tumefaciens by the freezing transformation method,and then transformed Columbia ecotype type Arabidopsis thaliana to get transgenic lines.Results showed that overexpression of maize MYB31 and MYB-IF35 could decrease the damage of chilling stress to photosynthetic reaction center,alleviate photoinhibition and increase the activity of antioxidant enzymes?SOD and APX?.As a result,overexpression of maize MYB31 and MYB-IF35 increased the resistance to chilling stress of Arabidopsis. |
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