| At present,there are more than 400000 known plant secondary metabolites,among which terpenoids mainly refer to compounds and their derivatives with isoprene unit(C5 unit)as the basic structural unit.Their skeletons are complex,diverse and have a wide range of biological activities.They play an important role in plant resistance to diseases and pests,stress,signal transduction and so on.In addition,plant metabonomics technology has developed rapidly in recent years,and has been widely used to study the overall changes and differences of plant metabolites under different genotypes and environments.In order to explore the types and contents of metabolites in cotton and further improve the insect resistance of cotton,the metabonomics method based on gas chromatography time of flight mass spectrometry(GC-TOF-MS)was used to analyze the representative metabolites in the seeds of Asian cotton,island cotton and upland cotton.Then,the key enzymes of trans inulin biosynthesis with insect resistance were introduced by using the sesquiterpenoid biosynthesis pathway of cotton itself,and the terpenoid metabolic pathway of cotton was reconstructed.In the future,we hope to obtain transgenic cotton lines rich in trans inulin.In this study,the metabolites in representative seeds of Asian cotton,island cotton and upland cotton were analyzed by GC-TOF-MS.Then,taking upland cotton as the chassis,using the sesquiterpenoid biosynthesis pathway of cotton itself,the key enzyme of trans inulin biosynthesis was introduced,and then the terpenoid metabolic pathway of cotton was reconstructed.In the future,we hope to get new insect resistant transgenic cotton lines rich in trans chrysanthemic acid.The results are as follows:1.Metabonomics of three cotton varieties(6 replicates)were analyzed by GC-TOFMS,and 706 metabolites were obtained.A total of 263 metabolites were screened and identified by univariate and multivariate statistical analysis,including amino acids,lipids,sugars,glycoalcohols and other nutrients.Compared with other cotton species,upland cotton contains more amino acids,sugars and other nutrients.The results showed that the content of tryptophan in upland cotton was higher than that in other two cotton varieties.2.Three key genes CDS,ADH2 and ALDH of trans inulin biosynthesis in pyrethrum were amplified and introduced into the modified plant expression vector p CAMBIA2300 to construct single gene and multi gene overexpression vectors p CAMBIA2300-CDS,p CAMBIA2300-ADH2-ALDH,p CAMBIA2300-CDS-ADH2-ALDH and p CAMBIA2300-RBCS-CDS The transient transformation in cotton leaves was verified by Agrobacterium tumefaciens injection.The results of qualitative analysis by GC-MS showed that pyrethrol could be detected in the transient expression leaves,which provided the basis for further stable genetic transformation.Extracted from transgenic cotton plants for fluorescence quantitative analysis.The results showed that compared with wild type,transgenic cotton plants with CDS gene had higher expression at RNA level.The results of GC-MS showed that the transgenic cotton plants could produce pyrethrol,which indicated that the new cotton lines could produce pyrethrol.3.Through hypocotyl transformation of cotton,31 samples of p CAMBIA2300-CDS transgenic cotton,3 samples of p CAMBIA2300-RBCS-CDS transgenic cotton.The experiment is in progress.The DNA of transgenic cotton plants was extracted and detected.There were 6 transgenic plants with p CAMBIA2300-RBCS-CDS and 2 transgenic plants with p CAMBIA2300-RBCS-CDS.The results showed that the foreign gene had been integrated into cotton genome.The results of Q-PCR analysis showed that the transgenic cotton plants with CDS gene expressed at the RNA level compared with the wild type.The results of GC-MS showed that there were 4 transgenic lines containing pyrethrol. |
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