| Pomegranate(Punica granatum L.)has a long history of cultivation and is widely planted in China.However,due to its poor cold resistance and susceptibility to freezing damage,it seriously restricts the development of China’s pomegranate industry.In view of this,studying the cold resistance mechanism of pomegranate under low temperature and exploring corresponding regulatory pathways is of great significance.This study compared the changes in various physiological indicators,including soluble protein,soluble sugar,relative electrical conductivity,proline,SOD,POD,MDA,and CAT,under different temperatures(4℃,0℃,-3℃,-6℃,-9℃,-12℃,and-15℃)after 24 hours of treatment on the annual branches of pomegranates from ‘Tunisia’,‘Zhongnonghong’,‘Hongshuangxi’,and ‘Qiancenghua’,the subordination function method was used to comprehensively evaluate the cold resistance of four pomegranate varieties.Then,through transcriptome sequencing,the differentially expressed genes involved in cold resistance regulation were analyzed,and the metabolic pathways involved by the differentially expressed genes were explored.On this basis,the key cold resistance genes were screened,and the CBF3 gene of pomegranate was cloned through molecular biotechnology,and the bioinformatics analysis was carried out to construct the expression vector,to provide theoretical basis for exploring the cold resistance molecular mechanism of pomegranate CBF gene in the future,the main research results are as follows:(1)The semi lethal temperature of ‘Qiancenghua’ is the lowest,reaching-6.94℃,while‘Hongshuangxi’ has the highest,reaching-2.27℃;Under low temperature stress,the relative conductivity,soluble sugar content,and POD activity of the branches of the four varieties showed an upward trend,while the soluble protein content,SOD activity,CAT activity,MDA content,and others showed a trend of first increasing and then decreasing,the overall content of proline shows a trend of first decreasing and then increasing.The comprehensive membership degree of ‘Qiancenghua’ is the highest at 0.56,while ‘Zhongnonghong’ has the lowest comprehensive membership degree at 0.45.The comprehensive evaluation of pomegranate using the membership function method shows that the order of cold resistance is: ‘Qiancenghua’> ‘Hongshuangxi’ > ‘Tunisia’ > ‘Zhongnonghong’.(2)Using Illumina Novaseq 6000 sequencing platform,the full-length transcriptome sequencing of ‘Tunisa’ and ‘Zhongnonghong’ soft seed pomegranate branches at 14℃ and0℃ was carried out,and 26582 expression genes were detected.GO function annotation analysis showed that 11538 and 10851 differential genes of ‘Tunisa’ and ‘Zhongnonghong’pomegranate varieties were annotated to a total of 50 functional groups,including molecular function There are three major categories of cellular components and biological processes.‘Tunisia’ and ‘Zhongnonghong’ have 4439 and 4179 differentially expressed genes respectively,which have been successfully annotated into KEGG database and enriched in five major categories of metabolism,biological system,environmental information processing,cell process and biological system.Among them,there are many differentially expressed genes in plant hormone signal transduction pathways such as abscisic acid,auxin and jasmonic acid,and in MAPK signal pathway.Key differentially expressed genes such as CBF1,CBF2,ICE1,COR and CBF3 have been screened,Fluorescence quantitative PCR validation revealed significant expression under low temperature stress.(3)Screening and cloning of candidate cold resistant gene CBF3 from pomegranate,successfully constructing an expression vector,and conducting bioinformatics analysis on it,it was found that its coding region sequence is 714 bp long,encoding 237 amino acids,and has a fixed AP2 conserved domain.Homology comparison showed that it has the highest homology with Arabidopsis CBF3. |
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