Cloning And Transformation Of Waxy Synthesis Genes From Karelinia Caspia

Global warming may aggravate the frequency and severity of extreme climate events.The increase of atmospheric CO₂concentration,temperature and the change of precipitation pattern caused by global warming may be important factors affecting plant growth and ecosystem structure and function(such as photosynthesis).Plant cuticular wax has the functions of water retention,reflecting ultraviolet radiation,high temperature resistance,freezing resistance and drought resistance,so as to enhance the ability of plants to adapt to and resist the external environment.The thick wax on the surface of leaves of desert plants Karelinia caspica may play an important role in coping with extreme temperature,drought,strong light and other stresses.In order to explore the effects of high temperature and drought stress on photosynthesis,the effects of leaf cuticle wax on photosynthesis were analyzed.At the same time,based on the research completed Karelinia caspica sticks at room temperature and high temperature contrast of RNA-seq data screening synthetic wax figure of the differentially expressed genes KcFAD2,Kc P450-77A,Kc HHT,using molecular cloning technology,for Karelinia caspica over the above-mentioned three genes of CDS,and on the above three genes encoding proteins bioinformatics and RT-PCR expression pattern analysis;The plant overexpression vector was constructed by Gateway technology,and Agrobacterium tumefaciens was used to mediate the transformation of Karelinia caspica,Nicotiana tabacum L.In this paper,the effects of high temperature stress on epidermal wax and photosynthesis in leaf organs of Karelinia caspica were analyzed,and the relationship between KcFAD2,Kc P450-77A,Kc HHT and plant high temperature tolerance was discussed,which provided a reference for the exploration and application of stress resistance gene resources of Karelinia caspica.The main research results are as follows:(1)Effects of epidermal wax accumulation on photosynthesis in leaf organs of desert plant Karelinia caspicaThe epidermal wax crystals of the leaf organs of wild desert Karelinia caspica are most closely distributed,which are mainly Rod,needle and flake.The are less wax on the leaf epidermis of artificial green space and indoor planting,and there are irregular sheet structure.The net photosynthetic rate and transpiration rate reached the maximum from 16:00 pm to 19:00 pm in the green room temperature environment,when the ambient temperature was 33?34?.In the same period of time,the net photosynthetic rate decreased by 88.2%in the desert environment,and the transpiration rate increased by 2.15 times.The concentration of intercellular CO₂and stomatal conductivity will reach the maximum from 11:00 am to 13:00am in the normal temperature environment of green space,when the ambient temperature is25?28?.In the same period of time,both the concentration of intercellular CO₂and stomatal conductivity show a significant downward trend are 62.9%and 93.72%respectively in the desert environment.The content of epidermal wax was significantly negatively correlated with the net photosynthetic rate and stomatal conductance of leaf organs,and the correlation coefficients were-0.84 and-0.69,respectively.There was a significant positive correlation between epidermal wax content and intercellular CO₂concentration,with a correlation coefficient of 0.53.There was no correlation between leaf epidermal wax content and transpiration rate.(2)Cloning and analysis of KcFAD2,Kc P450-77A and Kc HHT genes related to wax synthesis of Karelinia caspicaThe genes KcFAD2,Kc P450-77A and Kc HHT were cloned and analyzed by RT-PCR.The c DNA sequences and amino acid sequences of these genes were submitted to genebank,and the accession numbers were MV528401,MV528399 and MV528400,respectively.The full lengths of Kc P450-77A,Kc HHT and KcFAD2 were 1521 bp,1347 bp and 1152 bp,encoding 507,449 and 384 amino acids,respectively.Phylogenetic tree construction of the three genes showed that FAD2 gene similarity between KcFAD2 and Helianthus annuus was the highest(90.86%),the P450-77A gene similarity between Kc P450-77A and Helianthus annuus was the highest(90.32%),the similarity between Kc HHT and Lactuca sativa was the highest(86.50%).The expression patterns of the above three genes were analyzed by RT-PCR at 40?for 0 h,0.5 h,2 h,4 h,8 h,16 h,24 h and 48 h.It was found that the expression levels of the three genes were higher under normal conditions.With the prolongation of high temperature treatment time,the expression levels of the three genes showed a trend of decreasing first and then increasing.The expression patterns of above three genes were analyzed at different time points under the condition of high temperature,and it was found that the expression of the three genes showed a trend of high-low-high.Under 40?comdition,Kc P450-77A,Kc HHT and KcFAD2 genes had the highest expression at 8 h,4 h and 16 h,respectively.The results showed that the expression of above genes related to epidermal wax synthesis of Karelinia Caspia was affected by high temperature and showed a character of stress-induction-adaptation,which was consistent with the physiological response of plants under environmental stress.(3)Construction and genetic transformation of overexpression vectors of KcFAD2,Kc P450-77A and Kc HHTBased on the principle of homologous recombination,KcFAD2,Kc P450-77A and Kc HHT were constructed into the plant overexpression vector p K2GW7 by Gateway technology.p K2GW7-KcFAD2,p K2GW7-Kc P450-77A and p K2GW7-Kc HHT were successfully transferred into Agrobacterium tumefaciens LBA4404.A large number of callus and a small number of transgenic seedlings were obtained by leaf disc method,The preliminary genetic transformation of tobacco was completed,which laid a good foundation for the next step to verify the function of these genes.