Cold Resistance And Transcriptome Sequencing Analysis Of Tansgenic Arabidopsis Over-expressing CcCBFs

Low temperature stress is one of the important abiotic stresses affecting plant growth and development.The plant itself adapts to the external environment and forms a series of response mechanisms to the external environment.Among them,the CBF low temperature signal transcription factor pathway is resistant to plants.The cold process plays a decisive role.Cinnamomum camphora prefers a warm and humid climate,and its ability to adapt to low temperature is low.Generally,when the temperature is lower than 10?,growth is inhibited,growth is stopped at 5?,and freezing is likely to occur at 0? freezing point.When the tree grows,it can endure the short-term temperature of minus 10?.If it is at a low temperature for a long time,it may cause defoliation or even death of the tree.The long winter in the north of China has severely restricted its promotion and gardening applications in the north.This study is based on the previous laboratory study on CcCBFs(CcCBFa,CcCBFb,CcCBFc and CcCBFd)from C.camphora.In this study,wild-type,T2 transgenic CcCBFs gene Arabidopsis thaliana and T3 transgenic CcCBFs gene Arabidopsis were used as materials.Firstly,The physiological and biochemical indexes of the T2 and T3 generation of transgenic CcCBFs gene Arabidopsis under low temperature stress(4?)was detected.And the freezing resistance of T3 generation of transgenic CcCBFs gene Arabidopsis was tested by using-4? to simulate freezing injury.Then,the expression of the anti-oxidase gene and the late embryo rich gene of T3 generation of transgenic CcCBFs gene Arabidopsis after 6 days of low temperature stress were detected by q-PCR.From the above physiological indicators and the expression levels of stress-resistant genes,the CcCBFc gene with strong cold resistance was screened.Furthermore,transcriptome sequencing was carried out on T3 generation of transgenic CcCBFc gene Arabidopsis,and the differentially expressed genes were analyzed and studied.The downstream cold-resistant genes regulated by CcCBFc gene were identified,and the function ofCcCBFc gene was deduced.In this study,the transcriptome analysis of CcCBFc revealed the mechanism of CcCBFc exerting cold resistance in Arabidopsis,and the biological function of CcCBFc gene in resistance to low temperature stress was speculated.The main results of this study are as follows:1.The anti-reverse physiological indexes of transgenic CcCBFs gene Arabidopsis were detected and analyzed.The results showed that superoxide dismutase and proline content of the transgenic CcCBFa and CcCBFc genes Arabidopsis were significantly higher under low temperature stress.And overall higher than transgenic CcCBFb and CcCBFd genes Arabidopsis;malondialdehyde content was significantly lower than wild type and lower than CcCBFb and CcCBFd genes.It can be seen that the CcCBFa and CcCBFc genes are effective in enhancing the cold resistance of the T2 transgenic Arabidopsis.2.The stress resistance index of transgenic CcCBFs gene Arabidopsis was detected and analyzed under low temperature stress.The results showed that CcCBFc gene could significantly increase the content of peroxidase,superoxide dismutase,catalase and proline of transgenic Arabidopsis under low temperature stress and reduces the content of malondialdehyde.That is the CcCBFc gene can significantly improve the cold tolerance of the T3 transgenic Arabidopsis.3.Using-4? to simulate freezing damage,observe the phenotype of T3 transgenic CcCBFs gene Arabidopsis at this temperature.The results showed that the leaf area of wild-type Arabidopsis decreased and wilted after treatment at-4? for 14 days,and the plant tissues died.The leaf area of transgenic CcCBFa,CcCBFb and CcCBFd genes Arabidopsis decreased and wilted in varying degrees.The leaves of transgenic CcCBFc gene Arabidopsis were normally extended.The results showed that CcCBFc gene played a positive role in improving the freezing resistance of T3 transgenic CcCBFc gene Arabidopsis.4.Using q-PCR to detect the expression of the antioxidant enzyme gene and the late embryo-rich gene in the T3 generation of CcCBFs gene,the results showed that both the exogenous CcCBFc and CcCBFa genes could be up-regulated the expression levels of theoxide enzyme gene,the superoxide dismutase gene,the ascorbate peroxidase gene,and the late embryonic rich protein gene in transgenic Arabidopsis to improve the resistance to low temperature of T3 transgenic Arabidopsis.5.The transcriptome sequencing analysis showed that under positive conditions,there were 2335 differentially expressed genes between CcCBFc gene and wild-type Arabidopsis,and 1284 expression was up-regulated,of which 504 expression was significantly up-regulated.Under low temperature stress,the CcCBFc gene was transduced.There were 1125 differentially expressed genes in wild-type Arabidopsis,364 expressions were up-regulated,152 of them were significantly up-regulated;152 genes with significantly up-regulated expression were retrieved,and 51 genes related to cold resistance were obtained.6.Search for the up-regulation of 152 expression between CcCBFc gene and wild-type Arabidopsis under low temperature stress,and found that 6 kinds of cold-related transcriptions such as AP2/ERF,MYB,bHLH,bZIP,Zinc-finger and WRKY,which is involved in the transgenic CcCBFc gene Arabidopsis cold-resistant process.Among the transgenic CcCBFc gene Arabidopsis,the Zinc-finger family AT1G30135 gene and the MYB family AT1G18330 gene can be detected and expressed.Under low temperature stress,it is involved in jasmonic acid signaling pathway and ethylene signaling pathway.Therefore,it is speculated that the exogenous CcCBFc gene may regulate the constitutive expression of AT1G30135 and AT1G18330,participate in the regulation of jasmonic acid and ethylene signaling pathway,and regulate the synthesis of jasmonic acid and ethylene,improving the cold tolerance of transgenic Arabidopsis.7.In the differential gene analysis of 51 cold-tolerant genes between transgenic CcCBFc gene and wild-type Arabidopsis under low temperature stress,26 heat shock-related genes were retrieved,suggesting that these 26 heat shock-related genes may be associated with Cold-resistant correlation;20 heat shock-related genes in the transgenic CcCBFc gene Arabidopsis were detected and expressed in the non-cryogenic treatment.The results of differential gene cluster analysis showed that the transgenic CcCBFc gene Arabidopsis heat shock-related genes were relatively which expression level is higher thanthat of wild type.It is speculated that CcCBFc gene can up-regulate the expression level of heat shock related genes and finally play the function of cold resistance..