| The plant proteases are important enzymes that hydrolyze the peptide bonds in peptides.They play important roles in many life processes.Evidence has shown that plant serine carboxy peptide(SCP)proteins and SCP-like(SCPL)proteins with similar amino acid sequence to them are involved in the catalytic hydrolysis reactions and plant wound response.Moreover,SCP and SCP-like proteins also play a role in the synthesis of mustard acyl brmalic acid and brassinosteroid.However,whether these proteins modulate plant response to other adverse stresses is unclear.Previously,we obtained an Arabidopsis low potassium sensitive mutant named kl58(UUK+ lower mutantU58)by screening T-DNA mutant pool provided by Institute of Genetics and Development Biology of Chinese Academy of Sciences.The kl58 had small size and less lateral roots compared with wild-type(WT)plants under low K+ condition.Tail-PCR results suggest that a T-DNA reversely inserts the termination codon last base before the fourth bases of gene AT2G22920 in kl58.KL58 belongs to SCP family.RT-PCR analysis indicated that KL58 fails to express in kl58 seedlings.However,repeated experimental results revealed that the phenotype of kl58 under low K+conditions is unstable.To uncover the functions of KL58,we investigated the phenotype of kl58 under high iron,high zinc,low zinc or other stress conditions.The results showed that there is no significant difference between the mutant and WT plants during growth and development under these stresses.The KL58pro::GUS vector was constructed,transgenic plant lines of KL58pro::GUS were obtained,and GUS staining experiments were performed.GUS signal was strong in roots,weak in leaves in the seedling stage,and absent in the dried seeds,pods and inflorescence in KL58pro::GUS lines.The fusion vector of KL58 and green fluorescent protein(GFP)gene was generated,and subcellular localization of KL58 was studied.KL58 localizes in the cell membrane.To determine whether KL58 has serine carboxypeptidase activity,we built KL58-pET-28 a prokaryotic expression vector,and successfully induced the vector in bacteria and obtained the expressed protein of KL58.This will lay a foundation for further testing the serine carboxypeptidase activity of KL58 in Arabidopsis.Meanwhile,we investigated the effects of CRISPR/Cas9(Clustered regularly interspaced short palindromic repeats/CRISPR-associated 9)by silencing the NAD kinase 2 encoding gene NADK2 inArabidopsis thaliana.CRISPR/Cas9 system is another new genomic editing technology developed after the zinc finger nuclease and transcription activator-like effector nuclease technologies.It has the property of exactly and easily modifying genomic DNA after being used.At present,the technology has been applied more in human cells and medical science research.But,it has been used less in plant genomic editing.In this thesis,we try to edit gene NADK2 encoding NAD kinase 2,which catalyze the reaction for synthesis of NADP(H),by CRISPR/Cas9 in Arabidopsis.Vectors for editing NADK2 DNA sequence at two sites were constructed.Transgenic lines were generated by introducing the vectors into Arabidopsis wild type plants.The phenotypes of silencing NADK2 in transgenic plants were apparent,and the mutation rate by CRISPR/Cas9 was very high.We obtained 97 T1 transgenic lines.Fifty-seven lines had phenotypes similar to the T-DNA insertion mutant of NADK2,namely nadk2.Of which,17 lines were small in size with yellow leaves,40 lines were chimera with half yellow leave and half green leave.Other 40 lines had no clear phenotype.Statistically,the rate for T1 positive transgenic plants was 17.53%.The rate for chimera transgenic plants was 41.23%.In addition,the phenotypes and molecular identification results of T2 transgenic lines with two gRNA vector were the same as those of T1 lines.Taken together,these findings suggest that CRISPR/Cas9 technology can be used to silence NADK2 effectively in Arabidopsis. |
