| The Magnolia wufengensis is a new species of the Magnoliaceae and found in the natural secondary forest of Wufeng,Hubei in 2004.It belongs to the type of safflower tree and has greatly greening,ornamental and economic value.In recent years,new varieties of Magnolia wufengensis have been widely cultivated in the mountainous and plain areas of southern China,but when they were extended to northern areas of China,they have been trapped by overwintering injuries,which severely limited their scope of application.In order to enable the Magnolia wufengensis to successfully overwinter in northern China,in-depth exploration of the cold response mechanism of Magnolia wufengensis is an important part of its cold resistance research.In this study,Magnolia wufengensis was used as a material.Natural cooling,artificial low temperature and low temperature treatment after spraying with abscisic acid(ABA)were applied to analyze the physiological changes of Magnolia wufengensis under these three conditions.And then the molecular mechanism of cold response and ABA on cold-resistance improving in Magnolia wufengensis were further detected by using transcriptome method,and the functions of key cold resistance gene Mw CBF4 and its regulatory network were verified.The research results provide a theoretical basis for the cultivation of new Magnolia wufengensis varieties.The main results are as follows:1.The main reason for the poor cold resistance of Magnolia wufengensis in Beijing area was explored.By comparing the temperature in Beijing and Wufeng area from 2015 to 2017,it is found that the temperature in Beijing area from September to October is higher than that in Wufeng area(2.2 and1.2? higher,respectively),and the temperature in Beijing area in November is significantly lower than that in Wufeng area(5.4? lower).During this period,the content of chlorophyll,IAA,ZT and GA in the Magnolia wufengensis in Wufeng area decreased by 2.5%?26.2% higher than that in Beijing area.However,its accumulated osmotic adjustment substances,ABA and antioxidant enzyme activities were1.4% ? 66.0% higher than those in Beijing,and the malondialdehyde content and semi-lethal temperature of the plants were 0.8%?63.4% lower.Therefore,it is speculated that the primary reason for the poor cold resistance in Beijing area are that: the high temperature in Beijing from September to October delay the cold adaptation activating,coupled with the sudden temperature drop in November,leading to that Magnolia wufengensis don't have enough time to complete its cold-adapting process.2.The metabolic network of Magnolia wufengensis under cold stress was constructed.To compare the transcriptional changes of Magnolia wufengensis under room temperature and low temperature stress by Illumina transcriptome sequencing method.After De novo,gene function annotation and differential expression analysis,3910 differentially expressed genes were found.Then through GO and KEGG enrichment analysis,it was found that the differentially expressed genes were mainly enriched in functional categories such as membrane lipid structure,signal transduction,transcription factors,secondary metabolism,photosynthesis,and ROS clearance.Based on the expression of these genes,the metabolic network of Magnolia wufengensis under cold stress was constructed for the first time,providing a gene bank for new cold-resistant varieties cultivation.3.The molecular mechanism of exogenous ABA to improve the cold resistance of Magnolia wufengensis was analyzed.Through comparison of the cold response transcription changes of Magnolia wufengensis that caused by low temperature and exogenous ABA,it was found that exogenous ABA significantly changed the cold response process of Magnolia wufengensis.Only 1039 differentially expressed genes are common to both.Exogenous ABA caused the differential expression of 1500 specific genes.Through hierarchical expression clustering and GO enrichment analysis,it was found that exogenous ABA changed the hormone metabolism and signal transduction pathways in Magnolia wufengensis under low temperature stress,and significantly increased the expression level of 8transcription factor family genes under low temperature stress,promoted signal transduction,amino acid and sugar metabolism,organic acid biosynthesis and increased hydrolase activity.At the same time,it protected the photosynthesis of plants and accelerated the removal of harmful substances under low temperature stress.This laid a theoretical foundation for the cultivation of new cold-resistant varieties.4.The functions of the Mw CBF4 gene were verified.Based on the results of transcription sequencing,the sequence information of Mw CBF4 and its promoter was obtained by using Race experiment technology and genome walking technology.After amino acid sequence homology alignment and phylogenetic analysis,it was found that Mw CBF4 clustered in the A1 subgroup of CBF/DREB A-1 to A-6 subgroups,and it carried a conserved AP2/ERF DNA binding domain,with the CBF conserved sequences PKK/RPAGRx KFx ETRHP and DSAWR on both sides.In the AP2 domain,Mw CBF4 carries valine-14(V14)and glutamate-19(E19).Plant Care online analysis tool analysis found that the promoter sequence of Mw CBF4 gene contains 32 cis-acting elements.The Mw CBF4 gene was transferred into wild-type Arabidopsis thaliana by Agrobacterium-mediated dipping,and then the transgenic Arabidopsis thaliana was treated with low temperature and salt stress,it was found that the Mw CBF4 gene significantly improved the cold resistance and salt tolerance of Arabidopsis thaliana.In addition,it was also found that the function of the Mw CBF4 gene is closely related to ABA.5.The regulatory network of Mw CBF4 gene was constructed for the first time.Using the cis-acting element in the Mw CBF4 promoter as a bait,yeast library screening was performed through the yeast one-hybrid technology,and 10 upstream regulatory genes of Mw CBF4 were obtained,including 4positive regulatory genes(CES,ABF2/7/9),6 negative regulatory genes(MYB3/4,PIF1/3,PRR5,GA20ox1-B).And these 10 upstream regulatory genes can be matched with the GARE-motif,MYB,G-box and ABRE cis-acting element in Mw CBF4 promoter;using BD-Mw CBF4 as bait,the yeast library was screened by yeast two-hybrid technology,and 6 genes interacting with Mw CBF4 were obtained: SOD,Chlb 1,GFPT,Ph II,RSH2 and APY 1.Based on the above results,the regulatory network of Mw CBF4 gene was constructed.6.The main alternative splicing patterns and splicing sites of Mw RSH2 gene were clarified.It is determined that the Mw RSH2 gene interacting with Mw CBF4 has alternative splicing,which produces12 different copies through different splicing methods,and includes "GU-AG","AU-AU","AC-AG","AC-UG" and "UU-GG" five splicing sites.The alternative splicing patterns of Mw RSH2 gene mainly include normal splicing,exon skipping,intron non-splicing,exon skipping +3' and 5' cut position changes,cutpoint competition.One-to-one verification by yeast two-hybrid revealed that the Mw CBF4 gene only interacted with Splicing 1,Splicing 4,Splicing 5,Splicing 8,and Splicing 11 in the Mw RSH2 gene.In summary,this study explored the main reasons for the poor cold resistance of Magnolia wufengensis in Beijing,the metabolic network of Magnolia wufengensis under cold stress was revealed for the first time based on transcriptome data,and systematically analyzed the molecular mechanism of ABA improving cold resistance of Magnolia wufengensis,the molecular mechanism differences in cold resistance induced by ABA and non-freezing low temperature was determined.At the same time,the functions of Mw CBF4 and its regulatory network were verified for the first time.The main splicing sites and splicing patterns of 12 alternative splicing of Mw RSH2 gene were identified.The research results of this study provide a theoretical reference for exploring the mechanism of woody plants in response to low temperature stress from a comprehensive perspective,and lay a theoretical foundation for the cultivation of new varieties of Magnolia wufengensis. |
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