Study On Potato NAC Transcription Factors In Response To Low Temperature Stress

With the proposal of the national potato staple food strategy,winter leisure fields in southern China have become the most potential potato development area.Low temperature and rainy weather are the main limiting factors for potato production in the region.Studying the cold tolerance of potato materials and breeding cold-tolerant varieties is one of the important methods for the development of the potato planting industry in southern winter leisure fields.In this study,cold-tolerant materials were screened from 103 potato materials collected,which were verified by transcriptome sequencing and fluorescent quantitative PCR to screen and analyze transcription factors in response to low temperature.To study the function of potato NAC transcription factor in the response of potato to low temperature and low light stress by creating overexpression and interference transgenic materials.The main research results of this paper are as follows:1.Through electrical conductivity method and field natural frost method identification,using membership function method and cluster analysis method comprehensive analysis,Anti-10,Longshu No.5,21 parts of low temperature sensitive materials,GS393,BS214,16 parts of cold resistant materials,etc.were obtained.Meanwhile,the cold tolerance of potatoes from different sources was analyzed,which provides a certain reference for the directional collection of cold-resistant potato germplasm resources.2.Through transcriptome sequencing analysis of potato GS393 material under-3℃ low temperature stress there were 1692 differentially expressed genes,1436 up-regulated genes and 256 down-regulated genes.GO enrichment analysis found that the differentially expressed genes were mainly involved in phenylalanine ammonia lyase activity,cinnamic acid biosynthesis process,carboxylic acid metabolism process,carboxy-lyase activity,myo-inositol hexakisphosphate biosynthetic process,mandelonitrile lyase activity,dihydrokaempferol 4-reductase activity,acetyltransferase activity,guanosine tetraphosphate metabolic process,regulation of seed germination and response to gibberellin,etc.The KEGG metabolic pathway enrichment analysis found that the main pathways for differentially expressed genes are phenylpropanoid biosynthesis,phenylalanine metabolism,flavonoid biosynthesis,pyruvate metabolism,and plant hormone signal transduction.The St NAC1 and St NAC2 genes were determined for follow-up research through the screening of potato low temperature resistant differentially expressed transcription factors and partial fluorescence quantitative verification analysis.3.The expression of genes is spatial and temporal.The results of fluorescent quantitative PCR showed that the expression levels of St NAC1 in potato leaves,roots,stolon,stems,flowers,and tuber decreased in turn,the expression levels of St NAC2 in tuber,stolon,roots,leaves,flowers and stems decreased in turn.The results showed that under the low-temperature stress of 5℃,the expression levels of St NAC1 and St NAC2 genes increased significantly in a waves pattern,and the expression level of St NAC2 reached the highest value at 12 hours,which was 46.71 times that of the control.Under low-temperature stress of-3.5℃,the expression levels of St NAC1 and St NAC2 genes increased first and then decreased.The expression level was highest at 2 hours of stress,which was 3.24 and 3.58 times that of the control.Bioinformatics shows that both genes contain the functional domain of NAC transcription factors,have a mature protein tertiary structure,and have promoter core elements and promoter response cis elements such as hormone response.4.The functions of St NAC1 and St NAC2 of potato were studied by homologous recombination,and the overexpression and interference vectors were constructed with Solanum tuberosum(E3)as the transformation material.Through the verification of PCR and fluorescence quantitative PCR.We obtained four St NAC1 overexpression positive(OE-1)plants and two interference positive(Ri-1)plants;Six St NAC2 overexpression positive(OE-2)plants and five interference positive(Ri-2)plants.5.The phenotype of the transgenic materials showed that the growth cycle of the OE-St NAC2 transgenic materials lines was shortened,the number of tubers per plant of OE-St NAC1 and OE-St NAC2 lines increased.The growth cycle of Ri-St NAC is prolonged,the number of tubers in Ri-St NAC1 is the same as that of wild-type(E3),and the number of tubers in Ri-St NAC2 was significantly reduced.After 50 days of low temperature and low light treatment,the growth of OE-St NAC tissue culture seedlings are weaker than that of wild-type E3.The height of OE-1-3 plants is 0.28 times that of wild-type,and the height of OE-2-3 plants is 0.35 times that of wild-type.It showed that the internodes were shortened by low temperature and low light treatment;the growth of Ri-St NAC were better than that of wild type,the height of Ri-1-2 strain was 1.37 times that of wild type,and the height of Ri-2-4 strain was 1.13 times that of wild type.By observing the phenotype,we found that the OE-St NAC and Ri-St NAC strains have complementary phenotypes under the same treatment conditions.