| Hydrogen sulfide,considered as the third gasotrasmitter after nitric oxide and carbonic oxide,is playing a significant role in plant developmental processes and defenses.In recent years,with the increased environmental pollution,the growth and developmental processes of Chinese cabbage have been seriously inhibited.L-cysteine desulfhydrases（DES1 and LCD）are key enzymes to produce hydrogen sulfide.Integrating DES1 and LCD into cabbage genome can not only increase its stress resistance,but provide cabbage breeding with new germplasm.Besides,the clustered regularly interspaced short palindromic repeats（CRISPR）/Cas system known as the third generation of genome editing techniques has a higher gene-editing efficiency.Knocking out L-cysteine desulfhydrase genes by CRISPR/Cas could lay a theoretical foundation for the research of their functions and provide mutants for the latter study.On the basis of the existed transformation system,this study using inbred line ’aiqing 1’ as material optimized the seed sterilization time,microbial infection time and antibiotics concentration,so as to acquire an efficient transgenic system.At the same time,the overexpression vector XF-246-DES1,pCAM2300-LCD and knockout vector pYAO-LCD were constructed and the genetic transformation plants have been obtained successfully by the Agrobacterium-mediated transformation.The main research results are as follows:1.The construction of overexpression vector: p ET-28a-DES1 and XF-246 were digested by NcoⅠ-HF and Eco RⅠ-HF,and pET-28a-LCD and pCAM2300 were digested by BamHⅠ-HF and SalⅠ-HF.The digested products with same sticky end were linked directely and then transformed into E.Coli DH5α.Thus the overexpression vector XF-246-DES1 and pCAM2300-LCD were constructed which were verified by PCR amplification and restriction enzyme digestion.Afterward,the expression vectors were respectively transformed into Agrobacterium strain LBA4404.2.The construction of knockout mutate carrier: we designed and synthesized 20 bp of LCD gene which was linked to AtU6-26-sgRNA-SK vector,and then the recombinational vector AtU6-26-sgRNA-SK-LCD was obtained which was validated by PCR amplification and restriction enzymes digestion.Afterwards,the sgRNA cassette,the product of recombinant vector digested by NheⅠ-HFand SpeⅠ-HF,was linked to expression vector p YAO digested by SpeⅠ-HF and then transformed into E.Coli DH5α,thus we got the expression vector pYAO-LCD which was verified by enzyme digestion and transformed into Agrobacterium strain LBA4404.3.The optimization of the transformation system of Chinese cabbage: The results indicated that seeds disinfected with 0.1% HgCl2 for 5 min could maintain a higher rate of germination and it could also prevent the formation of endogenous bacteria simultaneously.When the explants were immersed in bacterium concentration of OD600=0.20.3 for 5 min and 7.5 mg·L-1 and 5.0 mg·L-1 were respectively resgarded as the resistant selection pressure for shoots and roots,the transformation rates were 10%,8% and 13.3% respectively of the overexpression vector XF246-DES1 and pCAM2300-LCD and the knockout vector pYAO-LCD.4.The detection of transgenetic plant: we detected the gene relative expression and H2 S contents of over-expressed plants by qRT-PCR and Four-channel radicals analyzer respectively,the results showed that the relative expression of DES1 and LCD and the H2 S contents in the plants were increased.The genome of transformed plants had been successfully edited by CRISPR/Cas9.Above all,the research constructed DES1 overexpression mutant,LCD overexpression mutant and LCD knockout mutant of Chinese cabbage by building their corresponding expression vectors and optimizing the genetic transformation system. |
