| Cannabis sativa L.,an annual herb of the genus Cannabis in the cannabinaceae,has been cultivated and utilized in China for thousands of years.The pharmaceutical industry hemp with CBD extraction as the main goal is an emerging industry in recent years.In the previous experiments,we found that due to the mechanical wounding to stems before harvest,the CBD content of hemp plants was higher than that of the control while the biological yield remained unchanged.This result can not only be suggested to improve the yield of CBD,but also has important value for studying the synthesis and regulation mechanism of secondary metabolites such as CBD.Therefore,this project applies mechanical wounding to the stems of female cannabis plants at flowering stage,using transcriptome sequencing,plant hormone content analysis,metabolome analysis and other methods,to explore the response regulation mechanism of stress defense system under mechanical wounding,and to establish regeneration and genetic transformation system for hemp.It lays a research foundation for understanding the stress defense mechanism of industrial hemp against abiotic stress and cultivating high-quality industrial hemp varieties.1.Mechanical wouding effects morphology and cannabinoid content of hempThe hemp plants of DMG 227 clonal seedling population were treated with stem puncture damage at the 7th week after flowering（one week before the normal harvest time）.The CBD contents of inflorescence at the 0d,2D,4D,7d and 10d after treatment were detected.It was found that the CBD contents reached the peak at the2D after treatment,and then decreased gradually,although the content increased in the later stage,However,it can not reach the peak level.The experiment was further verified in another clone seedling population of industrial hemp DMG 229.The contents of CBD and THC reached the peak at 1D and 2D after wounding,and then continued to decline,which is consistent with the experimental results of hemp material DMG 227.2.Differentially expressing genes increase after mechanical woundingTranscriptome sequencing analysis was performed on hemp samples at 0D,1D and2D after mechanical wounding treatment.A total of 2181 differentially expressing genes were screened in pair-comparation of the three samples,of which 518（336down regulated and 182 up regulated）and 1296（1031 down regulated and 265 up regulated）differentially expressing genes were found at 0d vs 1D and 0d vs 2D,respectively,The number of differentially expressing genes at 2D was 2.5 times that of the previous day,and the number of down-regulated genes was 3 times that of up-regulated genes.It shows that the number of differentially expressing genes increase sharply after wounding,and the down-regulated genes are significantly more than the up-regulated genes.It is speculated that in order to deal with the mechanical wounding of stems,plants have greatly reduced the gene expression to maintain the normal physiological and biochemical activities in the original growth state,so as to deal with the mechanical wounding.The enrichment analysis of all differentially expressing genes was carried through KEGG.Iit was found that the differentially expressing genes were mainly concentrated in the two biological pathways of primary metabolism and the synthesis of secondary metabolites,with a total of 75.4%.It is speculated that the primary and secondary metabolic pathways are closely related to the response of hemp to mechanical wounding.3.Endogenous hormones and metabolome change evidently after mechanical woundingTen plant hormones were detected in hemp samples at 0d,1D and 2D after mechanical damage treatment,and widely-targeted metabolomics was carried out.The results showed that the contents of JA,JA-ILE,SA and ABA changed significantly.A total of 23 categories and 995 metabolites were detected by widely-targeted metabolomics.432 primary metabolites and 563 secondary metabolites,including 77 differential metabolites were detected.Differential metabolites are mainly concentrated in metabolic pathways,flavonoid biosynthesis and biosynthesis of secondary metabolites.These biological processes of enrichment have a high correlation with the metabolic pathway of those in transcriptome analysis.4.The metabolic pathways plan important role after mechanical wounding stressMultiomics Joint Analysis showed that after mechanical wounding,the differential genes and metabolites with Pearson correlation coefficient greater than 0.8 in each control group were mainly enriched in the following metabolic pathways:metabolic pathways,biosynthesis of secondary metabolites,Plant hormone signal transduction,in which plant hormone signal transduction is the key research pathway in this paper.After mechanical wounding,the contents of JA,JA-ILE SA and ABA changed significantly,and many genes related to plant hormone signal transduction were also differentially expressed.In particular,the content of JA increased sharply at 2D after mechanical wounding,and the expression of signal transduction related genes also changed.It is speculated that JA,SA and ABA hormones and their downstream related genes play an extremely important role in the response and defense of hemp mechanical wounding.It is speculated that the stress defense mechanism of hemp is that after suffering mechanical wounding on the stem,the injury signal quickly passes through the cell membrane,and the content of plant hormones,reactive oxygen species and other related substances changes.As a signal,relevant transcription factors are activated and then regulates the expression of downstream genes.The expression of genes related to secondary metabolism is an important part of signal transduction,which leads to the increase of specific secondary metabolites in hemp plants,including tetrahydrocannabinol and cannabinoid.The metabolic pathways of other metabolites associated with the specific secondary metabolites of hemp,such as flavonoids and terpenes,have also changed accordingly,forming the material basis for coping with mechanical wounding.5.Genetic transformation of CBDAS gene of hempAs one of the recalcitrant plants,the in vitro regeneration system of hemp has always been the bottleneck of genetic transformation.In this study,a regeneration system using sterile seedling cotyledons as explants was established.The maximum regeneration frequency was 51.7%,and an average of 2-3 regenerated buds per explant could be obtained in a short time.In this method,MS medium containing 0.2mg/L TDZ and 0.05 mg/L NAA was used for bud induction.Although there are differences in regeneration efficiency due to different genotypes.In general,it is still an efficient method for the establishment of genetic transformation system,micro propagation and germplasm conservation.Based on the efficient in vitro regeneration system,we established and optimized the genetic transformation system of hemp by using the overexpression vector of CBDAS gene,which is the key gene in CBD synthesion.The effects of explant,Agrobacterium concentration and infection time were studied to optimize the system.The optimized transformation scheme used pre-cultured 1D cotyledons as explants,soaked with Agrobacterium with OD600 value of 0.6 for 20 min,and cultured for 3days in MS medium containing 100 mmol/L acetosyringone,250 mg/L cefotaxime,250 mg/L carboxybenzylpenicillin and 0.2 mg/L thiabenduron（TDZ）.Then the buds were transferred to the same medium as the selective culture media,and 35 mg/L hygromycin was added for resistance selection.The surviving buds were selected and transferred to 1/2 MS medium containing 0.1mg/l 3-Indolebutyricacid（IBA）for rooting.Gus histochemical analysis and PCR test confirmed the positive transformation.Although the regenerated plants with positive GUS expression were obtained and verified by PCR,most of these positive plants were chimeras,the insertion and expression of foreign genes only existed in some tissues,and the expression of foreign genes tended to disappear with generation reproduction.Therefore,the genetic transformation system of cannabis needs to be improved and optimized. |
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