| With the increasing of Greenhouse gas emission,the frequency of extreme weather events caused by global warming has increased markedly.Various kinds of extreme weather events,such as strong cold air,drought and floods have caused severe impacts on agriculture production.Maize is one of the most important cereal crops,forage crops and energy crops.Originated from tropical region,maize is a typical C4 plant in favor of heat and light,which demands a higher temperature conditions and is vulnerable to low temperature or freezing stress.During the maize production in China,the frost in spring and autumn usually cause severe impacts on the maize yield.Northeast China Region is the main producing area for maize in China.In Heilongjiang Province,maize planting area accounts for more than half of its total crop planting area annually.Therefore Heilongjiang Province has become the largest maize planting province in China.Moreover with the increasing comparative benefit of maize production,the maize planting area has reached the 6th accumulated temperature zone,which is usually unsuitable for the maize planting.Meanwhile,the freezing rain and snow weather events have become more frequent during the spring in Heilongjiang Province.Therefore the maize sowing time is prone to delay and maize seedlings easily suffer from freezing injuries during late spring coldness,affecting the normal growth and causing the yield loss.In conclusion,for maize production of Heilongjiang Province,it is of of profound theoretical and practice importance to study the freezing resistance of maize seedling,screen for freezing resistant maize inbred lines,investigate their physiological and biochemical mechanisms,uncover the freezing-resistance genes and carry out molecular assisted breeding.And in this study,thirty commonly used maize inbred lines in Heilongjiang Province were evaluated for freezing resistance.Combining the transcriptome sequencing with physiological analysis,the freezing responsive genes and related metabolic pathways of maize seedlings with different freezing resistance were compared.1.Effective evaluation method for maize seedling-stage freezing resistanceTo better simulate that weather conditions,we selected the maize seedlings of 3-leaf stage to be exposed to-1?for a period of time and then restored them to normal conditions for 3 days(25/20?,day/night).The seedling survival rates were recorded as the index of freezing resistance.To select the appropriate freezing treatment time for showing the difference between different inbred lines better,we set up 3 kinds of freezing time period,i.e.1 hour,3 hours and 5hours.And we found the seedling survival rates of 3 hours' freezing treatment range from 5.01%to 97.3%,with a normal distribution and best degree of differentiation.KR701 was selected asfreezing resistant inbred lines and Hei8834 as freezing sensitive inbred line.These two inbred lines were used for further study.2.Physiological mechanism of maize seedlings under freezing stressHei8834 showed apparent leaf withering and chlorosis.Microscopic observation after Trypan blue staining also showed the leaves of Hei8834 after freezing treatment were severely injured.To further study the physiological responses of these two inbred lines,we analyzed the common physiological indices,such as relative electric conductivity(REC),SOD activity,POD activity,soluble protein content and soluble sugar content.After freezing treatment,the REC of Hei8834 was 0.13 at 0.5h and 0.87 at 1h;the SOD activity increased to 57.3 U/mg at 1h and decreased afterwards;the POD activity increased to 53.7 U/mg at 0.5h and decreased afterwards;soluble protein and sugar content were both increased.For KR701,the REC was kept below 0.2after freezing treatment;the SOD activity increased to 80.8 U/mg at 2h and decreased slightly afterwards;the POD activity and soluble protein content increased all through the time point detected;the soluble sugar content increased until 2h and showed a slightly decreasing trend.Therefore,Hei8834 already showed sever freezing injuries at 1h after freezing treatment and its freezing resistance mechanisms failed.On the other hand,KR701 showed less freezing injuries within the 3 hours' freezing treatment and its freezing resistance mechanisms functioned effectively.But some physiological indices(SOD activity and soluble sugar)had showed a slightly decreasing tendency between 2h and 3h,suggesting the freezing resistance mechanisms could not function effectively if the freezing treatment time prolong.3.Transcriptome sequencing analysis of maize inbred lines with different levels of freezing resistanceThe mRNA of these two inbred lines before and after freezing treatment were extracted,used for cDNA library construction and next-generation sequencing.A total of 154.4 million sequences with 101 bp was obtained and 78.84% could be aligned to the maize reference genome Zea_mays_Ensembl_AGPv3.Cluster analysis and PCA results showed that samples of KR701 before and after freezing treatment were classified in the same group,samples of Hei8834 both before and after freezing treatment were classified together,suggesting that the primary factor underlying these different samples was inbred lines.A total of 19794 genes expressed in the 7samples of 4 different treatment.Among these expressed genes,360 genes were specifically expressed in KR701 after freezing treatment and 489 genes were specifically expressed in Hei8834 after freezing treatment.A total of 4550 genes were differentially expressed(DEGs)among 4 different treatment.Gene Ontology analysis were performed using these genes and significantly enriched GO terms were summarized.The freezing sensitive line specific genes after freezing treatment were specifically involved in some other biological process,such as response to low temperature and organic acid transport.Among those,ubiquitin ligase component was also enriched,suggesting much more severe freezing injuries in Hei8834.Also,the significant levels of same GO terms between thetwo inbred lines were different,suggesting the percent of genes involved in the same GO term were different.There were 439 DEGs for KR701 before and after freezing treatment and 852 DEGs for Hei8834.GO analysis showed that in biological process,the most significant GO term was freezing response for KR701 and low temperature response for Hei8834;in molecular function,the most significant GO term was binding for KR701 and protein tyrosine kinase activity for Hei8834;in cellular component,three GO terms significantly enriched in KR701 were all membrane related,while the most significant GO term was ubiquitin ligase component for Hei8834.Specific and shared DEGs were obtained by Venn diagram of the four comparison group.Among these genes,we screened for genes with differential responsive levels between inbred lines and performed Arabidopsis homology genes' function search.Genes involved in stress response and plant hormonal signaling were identified.Further functional analysis of these genes would deepen our understanding of the molecular mechanisms of maize seedlings' early freezing response and provide guidance for molecular assisted breeding of low temperature(freezing)resistant maize.At last,we performed quantitative real-time PCR(qRT-PCR)analysis of 30 selected genes.And the correlation coefficient between qRT-PCR and RNA-seq were 80% and 50% for freezing resistant inbred line and freezing sensitive inbred line respectively,indicating a good correlation between trancscriptome analysis results and qRT-PCR results. |
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