| Tobacco is an important leaf type industrial crop,and its leaf growth directly affects its economic value.Due to the three-dimensional climate,tobacco plants in higher altitude areas are easy to experience long-term low temperature at seedling stage,leading to early flowering of tobacco.Early flowering is related to the early differentiation of flower buds.Tobacco plants end their vegetative growth in advance,so that the number,quality and quality of leaves can not meet the production requirements.In previous studies,BRs signal pathway was closely linked to early flowering of tobacco through screening and analysis of gene expression profiles.In order to study the specific relationship between BR signal pathway and early flowering of tobacco,mutant plant B-2 of BRs signal receptor NtBRI1 gene has been obtained by Genome Editing,providing materials for subsequent experiments.In this experiment,based on previous studies,NtBRI1 overexpression vector was constructed,NtBRI1-OE overexpression transgenic plants were cultivated,and B-2 mutant and NtBRI1-OE transgenic plants were treated at different temperatures.Through observation,detection and analysis of the related phenotypes of agronomic traits and relative gene expression before and after treatment,the following conclusions are obtained:(1)Primers were designed according to the CDS sequence of tobacco NtBRI1 gene,and NtBRI1 gene sequence was successfully cloned.After sequencing,the consistency with Nicotiana tabacum in China Tobacco Genome Database reached 99.8%.The pC2301M1DPB-NtBRI1 overexpression vector was successfully constructed by using the cloned sequence,and the pC2301M1DPB-NtBRI1 overexpression vector was transferred to plant leaves by Agrobacterium infection method.After tissue culture,NtBRI1-OE transgenic plants were obtained by GUS staining,PCR identification and herbicide Basta identification.(2)After 6-leaf NtBRI1-OE plants and WT wild-type plants were treated at different temperatures,the related traits and gene expression were observed and detected.The results showed that the budding time of NtBRI1-OE overexpressed plants was 16 days earlier than that of wild-type plants at normal temperature,showing obvious early flowering phenomenon,but the phenotypes of NtBRI1-OE overexpressed plants were not significantly different from those of low-temperature treatment,indicating that plants had similar effects to low-temperature treatment after overexpressing NtBRI1 gene.The detection of relative gene expression also showed that overexpression of NtBRI1 gene eventually resulted in expression difference between NtFLC and NtLFY genes of transgenic plants and wild type,which may be the key factor leading to early budding of plants.This shows that BRs signal pathway is closely related to the phenomenon of early flowering at low temperature in tobacco.(3)Exogenous BR spraying experiments were carried out on B-2 Genome Editing mutant plants.Compared with the wild-type plants,it was found that the B-2 mutant plants could respond to exogenous BR as quickly as the wild-type plants,resulting in a sudden increase in the relative expression of NtBRI1 gene.However,the mutant plants could not smoothly transmit BRs signals downward,resulting in the expression of its downstream gene NtBSK3 much lower than that of the wild-type plants.This proved that the function of NtBRI1 gene in the B-2 mutant was deleted,which hindered the downward transmission of BR signal in BRs signal pathway to some extent.(4)After 6-leaf B-2 Genome Editing mutant plants and WT wild-type plants were treated at different temperatures,the related traits and gene expression were observed and detected.The results showed that after 10 days of treatment,the growth and development speed of B-2 mutant plants suddenly increased,which was significantly different from that of wild-type plants.This may be closely related to the effects of BR on cell division and cell elongation,but the specific molecular mechanism needs further study.At budding stage,the budding time of the two varieties under normal temperature treatment was not significantly different,but the budding time of B-2 mutant plants under low temperature treatment was 5 days later than that of wild-type plants,forming a very significant difference.After detecting the relative gene expression,it was found that the NtFLC gene expression was higher and the NtLFY gene expression was lower in B-2 mutant plants under low temperature treatment than in wild type plants,which would affect the flower bud differentiation of B-2 plants and eventually delay the flower bud differentiation time.That is to say,the loss of NtBRI1 gene function in B-2 mutant plant leads to the delay of budding time,which once again shows that BR signal pathway is closely related to tobacco low temperature early flowering. |
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