| Low temperature is an important factor affecting the growth and reproduction of tea trees.Extreme low temperature in winter and frost in early spring often cause low temperature damage to tea trees,affecting the quality of tea leaves and restricting the development of tea industry.During the process of cold acclimation,not only the physiological and biochemical processes of tea plant were changed,but also many specific proteins and m RNAs regulated by low temperature were induced to express.In recent years,it is a hot spot in the field of tea cultivation and breeding to study and understand the cold resistance mechanism of tea tree under low temperature stress,and to explore and utilize relevant cold resistance genes.In this study,the physiological and biochemical indexes of Yunwu Tribute Tea(Camellia sinensis(L.)Kuntze var.niaowangensis Q.H.Chen)under cold stress stress were measured to find out the critical temperature and critical time of stress,and the mechanism of Yunwu Tribute Tea defense against low temperature injury was discussed from the physiological perspective.Based on Illumina platform,transcriptome sequencing was performed to analyze the expression patterns of coldresistance related genes in Yunwu Tribute Tea and their correlation with physiological responses under cold stress stress.The function of polyphenol oxidase gene(Cs PPO)in tea plant was identified by transgenic technology to study the role of polyphenol oxidase gene on cold resistance of tea plant.It is of great theoretical and practical significance to study the cold resistance of tea tree for the utilization of germplasm resources,introduction,cold resistance breeding and the improvement of cold resistance cultivation technology of tea tree.The main research results are as follows:1.Physiological response of Yunwu Tribute Tea under cold stressUnder cold stress stress,the electrical conductivity and malondialdehyde(MDA)content of Yunwu Tribute Tea showed an upward trend with the extension of stress time,while the activities of antioxidant enzymes(SOD,POD,CAT),polyphenol oxidase(PPO)and osmotic regulatory substances(proline,soluble sugar,soluble protein)showed an upward trend first and then downward trend with the extension of stress time,and the maximum value basically appeared on the 3rd day of stress at 1 ℃.At the early stage of cold stress,Yunwu Tribute Tea can adapt to stress and reduce the damage caused by low temperature through its own regulation mechanism.With the extension of stress time,the protective effect of antioxidant enzymes and osmotic regulatory substances on membrane system and its regulation mechanism exceed a certain limit,resulting in metabolic disorder and irreversible damage.Under cold stress stress of 1 ℃ and 4 ℃,the content of abscisic acid(ABA)was significantly higher than the control,while the contents of indole acetic acid(IAA)and gibberellin(GA)were significantly lower than the control.With the increase of stress time,the contents of ABA and IAA were firstly increased and then decreased,while the content of GA was decreased.Under cold stress,Yunwu Tribute Tea could increase ABA growth inhibition substances,decrease IAA and GA growth promotion substances,and cope with low temperature by regulating endogenous hormone balance in plants.2.Transcriptome analysis of Yunwu Tribute Tea under cold stressIllumina Hiseq platform was used to sequence the transcriptome of Yunwu Tribute Tea hypothermic group(DW)and control group(CK)at 1 ℃ for 3 days.A total of 38.69 Gb of data were obtained,and 185,973 unigenes were assembled,with an average length of 992 bp and a functional annotation of about 61%.These unigenes are mainly enriched in Metabolic pathways and Biosynthesis of secondary metabolites under cold stress.The glutathione metabolic pathway,proline metabolic pathway,tyrosine metabolic pathway,carotenoid biosynthesis pathway and diterpenoid biosynthesis pathway related to the physiological indicators were selected to analysis,revealed 789 differentially expressed unigenes under cold stress,of which 366 unigenes were up-regulated and 423 unigenes were down-regulated.3.Correlation between expression level of cold-tolerance related genes and their response to physiological indicatorsFive cold-tolerance related genes with large differences in metabolic pathways related to early physiological indicators were analyzed.The expression pattern of glutathione oxidase(GPX),polyphenol oxidase(PPO),pyrrolin-5-carboxylate synthase(P5CS),9-cis-epoxide dioxygenase(NCED)and gibberellin 3β dioxygenase(GA3OX)were consistent with q RT-PCR and RNA-Seq.Under cold stress,the expression levels of GPX,P5 CS and NCED were significantly up-regulated.With the extension of stress time,the expression levels of GPX,P5 CS and NCED were firstly increased and then decreased,and their peak values were 2.6 times,2.4 times and 4.8times of the control,respectively.While,the the expression levels of PPO and GA3 OX were significantly down-regulated.With the extension of stress time,PPO expression level showed a trend of decreasing first and then increasing,and it was only11.1% of the control at 1 ℃ for 3 days,while GA3 OX expression level had no significant change.Under cold stress,the expression levels of GPX,PPO,P5 CS,GA3OX and NCED in Yunwu Tribute Tea were significantly positively correlated with POD activity,PPO activity,Pro content,GA content and ABA content,respectively.These results indicate that these genes play an important role in regulating their activity in response to physiological indicators.4.Cloning and functional identification of Yunwu Tribute Tea Cs PPO geneThe polyphenol oxidase gene Cs PPO of Yunwu Tribute Tea was cloned with a total length of 1740 bp,encoding 580 amino acids,and 96.72% homology with the polyphenol oxidase gene of Camellia sinensis.Cs PPO over-expression vector was constructed and overexpressed in tobacco by agrobacterium tumefaciens.Under 4 ℃cold stress stress,the expression level of Cs PPO increased first and then decreased,and reached the highest level on the 5th day of stress treatment,which was 3 times of the control group(wild type).To analysis the physiological characteristics of cold tolerance in transgenic tobacco showed that the cold tolerance of transgenic tobacco was higher than that of wild tobacco from phenotype under cold stress,and the relative conductivity and MDA content of transgenic tobacco were significantly lower than that of wild tobacco,while the activity of PPO was significantly higher than that of wild tobacco.These results indicated that transgenic tobacco could significantly reduce the damage degree of cell membrane,inhibit the lipid oxidation of cell membrane and improve the activity of polyphenol oxidase,thus significantly improving the cold tolerance of transgenic tobacco in low temperature environment. |
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