| Tobacco(Nicotiana tabacum L.)is an important cash crop in China and an important model plant for biological research.During the growth and development of tobacco,it likes temperature and does not tolerate low temperature,and low temperature is an important climatic factor restricting tobacco production.Plant phytochrome proteins,as two receptors of light and temperature,can sense changes in ambient light and temperature,and through the study of the low-temperature signaling mechanism of tobacco photopigment genes,biological technology can be used to mine anti-stress genes and explore their functions,which is of great significance for the selection and breeding of low-temperature resistant varieties.In this study,NtPhyA gene was cloned from tobacco,the gene sequence and protein characteristics were analyzed by bioinformatics,the transformation strain obtained by NtPhyA overexpression and knockout vector transformation tobacco was constructed,the function of the transformed strain under low temperature stress was explored,and the signaling mechanism of NtPhyA at low temperature was studied by transcriptome sequencing technology.The main findings are as follows:(1)NtPhyA bioinformatics analysis found that the gene coding region length was 2394 bp,encoded 797 amino acids,and had typical PHY family PAS,PHY and GAF motif special domains;NtPhyA protein is a non-secretory unstable hydrophilic protein without signal peptides;Subcellular localization predicts that the protein is localized in the cytoplasm and needs to be transported to the nucleus to regulate downstream genes.It was found that NtPhyA protein could be phosphorylated by kinases such as silk/threonine protein kinase and tyrosine protein kinase:cis-acting element analysis showed that NtPhyA could directly respond to low temperature signals and light signals,and could be activated by abscisic acid,methyl jasmonate,salicylic acid and other hormones as well as MYC and MYB transcription factors,while NtPhyA could be expressed in the whole plant and the expression amount between parts was not much different.It was shown that NtPhyA could sense cryogenic signals and may be involved in the signal response of tobacco hypothermia.(2)Under low temperature stress,the seed germination rate and germination time of NtPhyA knockout strains were better than those of wild type,and the degree of wilting was significantly lighter,while the overexpression strain was the opposite.The results of paraffin sections of various strains also showed that knockout NtPhyA could protect tobacco leaf cells from maintaining normal physiological structure at low temperatures,with a low degree of damage,and overexpressed strains were seriously damaged at low temperatures.Stomatal observation also showed that the stomatal structure of knockout strains was less damaged at low temperatures.From the phenotypic observations,knocking out NtPhyA increased the resistance of tobacco to low temperature stress.(3)After low temperature treatment,the hydrogen peroxide content and superoxide anion content of NtPhyA knockout strains were significantly lower than those of wild type,while the reactive oxygen species of overexpressed strains were significantly higher than those of wild type.Compared with the wild type,knockout strains had high SOD,POD,APX and CAT activities,lower relative conductivity and MDA content,and high proline content,while overexpressed strains showed the opposite.The net photosynthetic rate and Fv/Fm of knockout strains were also significantly higher than those of wild type,and the photoinhibition damage caused by low temperature was significantly lower than that of wild type and overexpressed strains,which could maintain normal photosynthesis at low temperature.The above results showed that NtPhyA cleared reactive oxygen species by increasing the activity of antioxidant enzymes at low temperature,maintaining cell membrane integrity and normal photosynthesis,indicating that NtPhyA is a negative regulator of tobacco low-temperature resistance.(4)Transcriptome sequencing analysis found that the differently expressed genes of transformed strains and wild types were mainly involved in biological processes such as metabolic process,cellular process,developmental process,bioregulatory process,stress response and cell tissue component by regulating transcriptional activity,catalytic activity,transport activity,binding and molecular function,affecting cellular components such as cell parts,cell membrane parts,organelle parts,protein-containing complexes and extracellular parts.These differently expressed genes are enriched in biological pathways such as secondary metabolite synthesis,environmental adaptation,signal transduction,amino acid metabolism,lipid metabolism,carbohydrate metabolism,membrane transport,and energy metabolism.The results show that NtPhyA can respond to low temperature,amplify signal transmission,activate downstream related genes to generate corresponding metabolites,and improve the hypothermia resistance of tobacco.(5)Among the differently expressed genes,the Nitab4.5＿0001286g0040 gene and the Nitab4.5＿0000108g0090 gene will participate in the low-temperature signaling mechanism by transmitting low-temperature signals and phosphorylating the corresponding genes in the signal transduction pathway;Nitab4.5＿0000269g0160 genes and Nitab4.5＿0000508g0210 genes participate in the resistance to low temperature by regulating metabolic pathway related genes such as secondary metabolite synthesis and amino acid metabolism.Nitab4.5＿0006960g0010genes and Nitab4.5＿0009134g0010 genes can regulate enzyme activities in the antioxidant system and protect the normal physiological functions of cells from being damaged at low temperatures.Nitab4.5＿0000134g0030 genes prevent damage to photosystem Ⅱ caused by low temperatures in photosynthesis,resulting in photosuppression.The above differentially expressed genes may be downstream target genes of NtPhyA in tobacco cryogenic signaling.(6)The transcription factor analysis of differentially expressed genes found that NtPhyA could conduct and amplify low-temperature signals by influencing transcription factors such as SPB transcription factor,ERF transcription factor,MYB transcription factor,HB transcription factor,bHLH transcription factor,WRKY transcription factor,MIKC transcription factor,B-3transcription factor and M＿type transcription factor,and activate downstream cold response genes.In summary,under low temperature stress,NtPhyA senses the low temperature signal,amplifies the low temperature signal transmission through a series of transcription factors,activates the gene regulation of reactive oxygen species and antioxidant enzyme activities in the antioxidant system of downstream secondary metabolite synthesis,environmental adaptation,signal transduction,amino acid metabolism,lipid metabolism,carbohydrate metabolism,membrane transport,energy metabolism and other related biological pathways,maintains the integrity of cell membrane and the normal operation of light and system,and protects the normal physiological function and cell structure of the tobacco plant at low temperature.Improved resistance to low temperatures. |
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