| Temperature stress seriously affects the growth and development of tobacco,causes the loss of yield and quality,and which endangers the safety and stability of tobacco production.It is significance to mine and use temperature stress tolerance genes to improve the adaptability of crops to temperature stress.In this study,the expression pattern of NtHSP70-8 gene under different tissues,hormones and abiotic stresses were analyzed on the basis of obtaining the over expression transgenic lines of tobacco,and NtHSP70-8 gene was edited by CRISPR / Cas9 gene editing technology in tobacco.The difference of response to high temperature and low temperature stress between over expression and gene editing of NtHSP70-8 plants at seedling stage was studied,and the function of NtHSP70-8 in tobacco temperature stress was also dissected.It can provide provide a basis for further studying the mechanism of tobacco response to temperature stress and improving the tolerance of tobacco varieties to temperature stress.The main findings are as follows:The expression pattern of NtHSP70-8 in different tissues and organs of common tobacco K326 and under different abiotic stresses was analyzed by q PCR.It was found that the NtHSP70-8 gene was expressed in different tissues and predominant expression in little corolla and leaves of tobacco,and its expression was induced by external hormones(GA3 100 μM,ABA 100 μM),high temperature(45℃),low temperature(4 ℃),and drought(PEG 20%)at seedling stage.The different T0 generation NtHSP70-8 gene ko strains were created by CRISPR/Cas9 technology.Then,three ko strains were acquired by self-crossing and sequencing different edited plant,ko-1 with a single base T insert at the 408 th position,ko-1 with a single base G insert at the 394 th position,ko-3 with T absent at the 391 st position along with base A and base G exchange at the 393 th position.The 7-week-old overexpression strains seedlings(OE-1,OE-2,OE-3)and gene editing strains seedlings(ko-1,ko-2,ko-3)with wild-type tobacco K326(WT)as control were treated with high temperature stress(45 °C for 56 h).It was found that the wilting degree of the three OE strains was light,and the leaf chlorophyll content was significantly higher than that of WT and ko strains,showing relatively high temperature resistance.The wilting degree of ko strains was the heaviest,and the leaf chlorophyll content was significantly lower than that of WT.Further study found that under high temperature stress,the leaf relative conductivity,MDA and H2O2 contents of OE strains were significantly lower than those of WT and ko strains,while the antioxidant enzyme activity and antioxidant enzyme related gene transcription level were significantly higher than those of WT and ko strains,showing strong leaf antioxidant enzyme activity.Under high temperature stress,the expression levels of high temperature,defense and ABA related genes in leaves of OE strain were significantly higher than those of WT and ko strain.The expression levels of NtHSP90,NtHSP101,Nt AREB and Nt NCED genes of OE-3 were 1.8 times,2 times,2.1 times and 2.7 times higher than those of WT respectively,while the expression level of related genes of ko strain was significantly lower than that of WT.The 6-week-old different strains tobacco seedlings(WT,OE,KO)were also treated with low temperature stress(4 °C for 12 h).It was found that the wilting degree of the three ko strains was lighter,and the leaf chlorophyll content was significantly higher than that of the WT and OE strains,meaning relatively high low temperature tolerance.Furthermore,compared with WT strain,the content of MDA,H2O2 as well as relative conductivity in ko strains were obviously decrease at low temperature stress,while antioxidant enzyme activity and antioxidant enzyme-related gene transcription levels were significantly increased.Under low temperature stress,genes associated with defense and low temperature in ko strains were also higher expression levels than WT and OE strains.In conclusion,NtHSP70-8 has a positive regulatory effect on tobacco high temperature stress and a negative regulatory effect on tobacco low temperature stress. |
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