| As global warming intensifies,the growth and development of plants are affected to varying degrees,resulting in crop yield reduction.Therefore,high temperature has become an important abiotic stress affecting crop production.The response and regulation mechanisms of plants in the process of resistance and adaptation to high temperature has become to the trend of current research in plant science.Tomato（Solanum lycopersicum）is one of the most widely cultivated vegetables in the world and also one of the most important model plants for the research of gene function of plants.Using tomato to reveal the high temperature resistance mechanism of plants can lay a certain foundation for the cultivation of new high temperature resistant varieties.The bZIP transcription factors are a type of transcription factors that are ubiquitous in plants.A large number of studies have shown that bZIP transcription factors are involved in various biological processes,such as growth regulation,the adaption to the abiotic and biotic stress,and so on.However,the research on the tomato bZIP transcription factors which involved in the regulation of heat stress is still scarce.It is of great significance to explore the expression and regulation of bZIP family genes in tomato under heat stress,and to analyze their molecular mechanism.In our previous study,we found the tomato bZIP transcription factor,SlbZIP2,which was involved in the heat stress base on the gene chip screening and bioinformatics analysis.It may participate in the tomato heat stress response.In this study,the SlbZIP2 was colned and analysis by the bioinformatics.The gene expression pattern and regulator analysis were tested by the q RT-PCR.The progeny of SlbZIP2 OE transgenic plants was observed under heat stress.The relevant physiological indicators and the expression of relative genes were determined after heat stress to confirmed the function of SlbZIP2 gene in the heat adaptation process of tomato.The main results are as follows:1.Gene cloning and bioinformatics analysis of SlbZIP2 geneThe target fragment of the SlbZIP2 gene of 621 bp size was obtained using high-fidelity enzyme and specific primers,and the c DNA of wild-type tomato AC++is used as a template.SlbZIP2 gene including 441 bp CDS sequence base on sequencing results.It is predicted that this gene encodes 146 amino acids,and its molecular weight is 16.69 KD.It is speculated that this protein is an unstable hydrophilic protein.it’s the promoter region analysis revealed that there were38 types of cis-acting elements.Most of the cis-acting elements were related to abscisic acid reaction,light response,gibberellin reaction and other functions.It was found that SlbZIP2 shares two common conserved domains which are conserved domains of the bZIP family transcription factor.2.The expression pattern analysis of SlbZIP2 gene expression patternThe RNA samples including root,stem,young leaf,mature leaf,senile leaf,flower,sepal and fruit from wild type tomato AC++were collected,and the expression of SlbZIP2 gene was analyzed by q RT-PCR.The results showed that the gene is expressed in various parts of wild-type tomatoes.The expression of SlbZIP2 gene is higher in roots and flowers rather than other tissues.And during fruit ripening,the gene expression is relatively stable,and there is no obvious increase or decrease with the ripening period.3.The expression pattern analysis of SlbZIP2 gene under abiotic stressesThe wild-type tomato AC++RNA samples were collected from root,stem,and leaf which were treated with various abiotic stresses.The q RT-PCR were used to analyzed for the expression of SlbZIP2 gene in different tissues under various abiotic stress treatments.The results showed that heat stress can significantly induce the expression of SlbZIP2 gene,and the gene expression was increased with the higher temperature and sustained stress.Drought stress can induce the expression of SlbZIP2 gene,and the expression level also reduce because of watering to ease drought.salt stress is a factor that induces the expression of the SlbZIP2 gene,and the gene expression was increased with the salt concentration.However,the expression of the SlbZIP2 gene has no significant changes compared with the control treatment under cold stress treatment.Cold stress may not be the factor that induces SlbZIP2 gene expression.4.The vector construction and tomato transformationSlbZIP2 gene and p VCT2024 vector were digested with Bam H I and Sac I,the fragments were recovered and ligated with T4 ligase.And the expression vector p VCT2024-SlbZIP2 was successfully constructed.The target fragment of mutation sites was obtained by PCR amplification,the p KSE401 vector was digested by Bsa I enzyme and then recovered.The target fragment and vector fragment Were linked with T4 DNA ligase,then transformed into E.coli.The CRISPR/Cas9silent expression vector was successfully constructed.The positive transgenic seedlings were obtained through the Agrobacterium-mediated method using the overexpression vector.The expression level of SlbZIP2 gene in the transgenic seedlings was analyzed by q RT-PCR.Six overexpression lines were successfully obtained OE-4,OE-6,OE-7,OE-8,OE-11 and OE-12.While,the CRISPR/Cas9 silent expression vector was transferred into AC++and positive seedlings were obtained.Unfortunately,the gene editing silenced lines were not obtained.5.Phenotype observation of transgenic tomatoes heat stress treatmentThree lines（OE-6,OE-11 and OE-12）with the highest gene expression were selected.The three SlbZIP2 gene overexpression lines tomato and wild-type tomato AC++were simultaneously treated at 42°C and 25°C.After 3 days of heat stress treatment,the leaves of the wild-type tomato suffered severe water loss,and the stems were severely wilted.However,the transgenic tomato leaves were only slightly wilted.The damage caused by heat stress to wild-type tomatoes is significantly stronger than that of transgenic tomatoes.It indicating that transgenic plants are more resistant to heat stress than wild-type tomatoes.Therefore,overexpression of SlbZIP2 gene enhances the heat resistance of tomato.6.Determination of relevant physiological indexes after heat stressThe tomato samples were collected before and after the heat stress treatment.The relative conductivity,chlorophyll content,proline content and malondialdehyde content were measured.The results showed that the relative conductivity of the leaves of overexpressed plants was significantly lower than that of wild-type plants after heat stress.And the chlorophyll content and malondialdehyde content were also lower than that of wild-type plants,and the content of proline was higher than that of wild-type plants.These results reflect that the Transgenic plants suffer less damage under heat stress,indicating that the overexpression of the SlbZIP2 gene enhances the heat tolerance of tomatoes.7.Relative gene expression after heat stressAfter heat stress,the samples of transgenic tomato and wild type tomato were collected,and the expression of SlbZIP2 high temperature related genes and ABA signal pathway related genes were analyzed.The results showed that under heat stress,the expression of Hsf A1,Hsf A2,Sl SIZ1,Sl AREB1,Sl PP2C2,Sl TASI4 and Sl NCED genes had significant changes.It was speculated that SlbZIP2 effected the resistance of tomato to high temperature stress by regulating the expression of related genes,and might participate in ABA signaling pathway. |
