CDNA Cloning And Construction Of CRISPR-cas9 Editing Vector Of Five Differentially Expressed Genes Induced By Hormone ?1 In Phytophthora

Phytophthora is a class of destructive plant pathogens that affects a wide range and causes serious economic losses.Its sexual reproduction is very important for the continuation and evolution of species,but the molecular mechanism is still unclear.In our previous work,Proteomics method was used to detect the protein expression differences of the isolate P7723(A2 mating type)of Phytophthora infestans induced by hormone al and a batch of differentially expressed proteins were obtained.In this study,the gene PITG-03644,PITG-04663,PITG-06415,PITG-13014,and PITG-17706 were chosed as the research objects.The protein coding regions of cDNA of five genes were cloned and the sequences were analyzed.The CRISPR-cas9 gene editing vectors of these genes were constructed.Our work lays a foundation for further study on the function of the five genes and the application of CRISPR-cas9 gene editing technology in P.infestans.The results showed that the sequence of the coding region of the cDNA of PITG-03644 of the isolate P7723 of P.infestans was 609bp long and the encoded amino acids were 202,which was 100%similar to the nucleotide and amino acid sequences published on NCBI.The coding region of the cDNA of PITG-04663 was 1760bp long and the encoded amino acids were 587,which was 100%similar to the nucleotide and amino acid sequences published on NCBI.The coding region of the cDNA of PITG-06415 was 2121bp long and the encoded amino acids were 706,which was 100%similar to the nucleotide and amino acid sequences published on NCBI.The coding region of the cDNA of PITG-13014 was 1182bp long and the encoded amino acids were 393,which was 100%similar to the nucleotide and amino acid sequences published on NCBI.The coding region of the cDNA of PITG-17706 was 2337bp long and the encoded amino acids were 778,which was 99.57%and 99.61%respectively similar to the nucleotide and amino acid sequences published by NCBI.Compared with the published nucleotide sequence,the coding region of the cDNA of PITG-17706 cloned in this study has the insertion mutation of 9 bases TTCGTCAGC between 878 to 886bp,resulting in the addition of 3 amino acids SAS in the amino acid sequence.In addition,compared with the published nucleotide sequences,the sequence cloned in this study was mutated from C to T at the 29th base,but the mutation did not cause any changes in amino acids.The three-dimensional structure of the protein predicted based on this sequence is exactly the same as that predicted based on the NCBI sequence,so it is speculated that there is no difference in their functions.Bioinformatics analysis showed that the protein encoded by PITG-03644 was a nucleoprotein,possibly a hydrophobic protein with typical Rab family and P-loop_NTPase superfamily domain.The protein encoded by PITG-04663 was a nucleoprotein,possibly a hydrophobic protein with typical bZIP domain,SRPBCC superfamily domain and Med15 domain.The protein encoded by PITG-06415 was a cytoplasmic protein,possibly a hydrophobic protein with a typical PTZ00272 domain.The protein encoded by PITG-13014 was a cytoplasmic protein,possibly a hydrophobic protein with a typical APP_MetAP family domain.The protein encoded by PITG-17706 was a nucleoprotein,a hydrophilic protein with a typical bZIP domain.None of these proteins contained significant transmembrane regions.Two sgRNA targets were designed for the protein coding region of each gene,and they were successfully cloned into the gene editing vector pYF515.A total of 10 CRISPR-cas9 gene editing vectors of 5 genes were successfully constructed.