| Fatty acid biosynthesis systems are an important factor in plants' resistance to external stresses,and plants can respond to various adversities by regulating their own fatty acid synthesis.Temperature is one of the main environmental factors affecting plant distribution.High temperature not only affects the growth and development of plants,but also seriously affects plant reproduction.Plant epidermis wax not only plays a role in water retention,cleaning,prevention of other pathogens and external damage,but also reduces ultraviolet radiation,resists high temperature,freezing and other external environment stress.In order to elucidate the relationship between epidermal wax synthesis and high temperature resistance of Karelinia caspia.In this study,we analyzed the fatty acid biosynthesis transcriptome and its differentially expressed genes in the surface epidermis waxy and high temperature conditions of desert plant Karelinia capsia under different conditions,predict gene function and constructing super expression vector of the key enzyme KcFabZ gene for the biosynthesis of fatty acids,the main results are as follows:(1)Extraction and analysis for epidermal wax of Karelinia caspiaAccording to tthe orthogonal experimental design,analysis of variance and electron microscopy of the waxy epidermis of the Karelinia caspia leaves showed that,the extraction temperature has the greatest influence on the extraction results,followed by the extraction time,the extraction liquid and the extraction material.According to the coverage ratio of leaf wax of each treatment leaf,the degree of leaf wrinkles and the wax extraction efficiency around the pores,the extraction condition was the best for the extraction of chloroform at 60°C.The extracted wax was analyzed and found to be the main component of the alkane.Under the high temperature treatment,the total alkane content increased with the increase of the high temperature treatment time,and the total alkane content was the highest after 24 hours of treatment,about 6 times of untreated materials,but after 72 hours of high temperature treatment,the total content of alkanes suddenly decreased,but still more than twice the amount of untreated materials,It is speculated that it is a mechanism for self-protection of Karelinia caspia to regulate the wax content of leaf epidermis under long time high temperature stress.Comparing the wax quality of Karelinia caspia leaves in different kinds of environment,it is found that the total content of alkanes in artificial green space is 10 times times that of indoor culture materials,the total content content of alkanes in field desert materials is 18 times times that of indoor culture materials,In the comparison of the total alkane content of the materials in the artificial green space and the field desert environment,it is found that the content of alkanes in two kinds of field environment is higher,and the content decreases when it is in a long period of high temperature,which further proves that the Karelinia caspia adopts the content of regulating the wax of the epidermis to resist high temperature stress.(2)Analysis of fatty acid biosynthesis transcription group and expression patterns of related genes at high temperature of Karelinia caspiaA total of 11 fatty acid synthesis genes were differentially expressed in the normal temperature-high temperature RNA-seq transcriptome of Karelinia caspia,which were mainly divided into three categories.The first one was the acetyl-CoA carboxylase(ACC)gene family,which mainly catalyzes acetyl-CoA.Is the rate-limiting enzyme that controls fatty acid biosynthesis;the second is the fatty acid synthase(Fab)family gene,which is responsible for the reduction and dehydration of the carbon chain;the third is the fatty acyl-ACP thioesterase responsible for terminating the carbon chain elongation.(Fat)family genes,through homology analysis,these three types of genes and Helianthus annuus,Lactuca sativaLinn,Cynara cardunculus and other species of higher homology,both reached more than 60% and It has the highest affinity with Lactuca sativaLinn;in the analysis of the expression patterns of some genes,it is found that each gene is differentially expressed in high temperature treatment,and the trend is slightly different: under high temperature treatment,the gene KcACC shows a trend of decrease-rise-reduction.The genes KcFabZ and KcFatB showed a trend of decrease-rise-low-rise-increased,and the gene KcFabG5 showed a continuous increase-decrease trend,and the gene KcFabG6 showed a continuous increase-Low-increase-decrease-increasing trend;Through the interaction analysis of the differentially expressed genes and proteins associated with waxy pathways,it was found that the fatty acid biosynthesis genes interacted with their direct proteins.(3)Functional prediction of KcFabZ gene and construction of overexpression vectorThe KcFabZ gene CDS is 669 bp in length and is located in the chloroplast.The chloroplast is a kind of plastid.The first step of wax synthesis is to synthesize fatty acids of length C16~C18 in the plastid,The second step is in the endoplasmic reticulum and Long-chain fatty acids extend carbon chains,and the third ultra-long-chain fatty acids form alcohols,esters,and alkanes under the modification of the endoplasmic reticulum.Therefore,it is speculated that this gene is involved in leaf surface wax accumulationof Karelinia caspia;phylogenetic results show KcFabZ The gene homology with the Lactuca sativaLinn reached 89%;according to the conserved domain and 3D structure in NCBI,KcFabZ is a hot_dog superfamily functional gene with a typical highly conserved ?+?'hot_dog' fold;real-time PCR The results showed that the expression of KcFabZ increased first and then decreased with the treatment time under high temperature treatment.The expression reached the maximum at 8 h after high temperature treatment,which was 19.54 times of the control.The results showed that the expression of KcFaZ gene was related to high temperature treatment.The results of this study show that the epidermal wax of Karelinia caspia is extracted as the best extraction method under the 60 s heating of trichloromethane.The main components of the epidermis wax of Karelinia caspia are alkanes,and the Karelinia caspia is resistant to high temperature by regulating the wax content of the epidermis under long-term high temperature stress.The only dehydrated enzyme gene in the biosynthesis pathway of cloned fatty acids KcFabZ also showed different expressions under high temperature treatment,which provided a theoretical basis for continuing to explore the response mechanism of Karelinia caspia under high temperature stress and Provide theoretical basis and genetic resources for high temperature breeding of Cotton,etc.cash crops and other economic crops. |
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