| Sterol 14a-demethylase(CYP51)belongs to the cytochrome P450 superfamily.It plays an important role in the synthesis of sterols and cellulose in organisms,as well as in the synthesis of plant hormones such as brassinolide,and is also a target enzyme for a variety of triazole fungicides.The previous studies mainly focused on the fungal disease control by fungicides,little is known about the function of plant CYP51.Based on the cloning of two wheat CYP51 genes,the structural characteristics,expression characteristics and possible functions of CYP51 gene were studied.The main research contents are as follows:1.The results of germination test of propionibacteriazole seed dressing treatment showed that the suitable concentration of seed dressing had a certain promoting effect on wheat growth,and increased the biomass of wheat seedlings in the treatment group,and the inhibition of high concentration treatment was significant;Prothionazole treatment increase the water content,soluble sugar and chlorophyll content of wheat,and had a positive effect on the biomass accumulation of wheat seedlings,decrease cellulose content,and water loss was significantly greater in leaves than that in control.2.Two CYP51 family genes,Ta CYP51H6-D and Ta CYP51H9-A were cloned by PCR.The genomic sequence of Ta CYP51H6-D is 2994 bp contains two introns.The c DNA of Ta CYP51H6-D is 1849 bp,including a 190-bp 5' non coding region,183-bp 3' non coding region,and a 1476-bp open reading frame encoding 491 amino acids,the molecular weight is about 55.76 k D,and the isoelectric point is 9.22,belong to alkaline protein.The genomic sequence of Ta CYP51H9-A is 2269 bp containing two introns.The c DNA of Ta CYP51H9-A is 1867 bp,the length of 5' non-coding region is 137 bp,the 3' non-coding region is 203 bp,the open reading frame encodes 508 amino acids,the molecular weight is about 57.01 k D,and the isoelectric point is 9.89,which belongs to alkaline protein.Protein sequence analysis showed that both Ta CYP51H9-A and Ta CYP51H6-D contained a typical heme iron binding domain of P45 family members.The genetic evolution tree shown that the above two genes are similar to the rice CYP51 H subfamily members with similarity and similar structure,so they are classified as CYP51 H subfamily.3.Genome editing vectors for 10 different CYP51 family members in rice were constructed by CRISPR/Cas9 system.Transgenic lines were obtained in all 9 genes except Os CYP51H8 gene by Agrobacterium-mediated genetic transformation.Using CRISPR–Cas9genome editing,knockout homozygous mutants(CYP51H6GMO-1)of the Os CYP51H6 gene were generated,which one base was inserted at position 260 in the CYP51H6 coding region.The germination experiment of CYP51H6GMO-1 were performed,and the results showed that the germination rate of CYP51H6GMO-1 was significantly lower than that of the wild type control,and in vitro addition of 100 n M concentration of brassinolide can partially restore the germination rate of the edited line,and the recovery rate is also enhanced with the increase of the added concentration,with a certain dose effect,indicating that Os CYP51H6 is closely related to BR synthesis or signal transduction pathway.However,under high-concentration BR treatment conditions,the germination rate of the edited lines is inhibited and significantly slower than the control,which suggests that Os CYP51H6 functions are diverse,and the specific functions need to be studied;Multiple edited plants of CYP51 genes showed decreased fertility,abnormal tillering and plant height when planted in controlled greenhouse.The specific phenotype,function and internal mechanism need to be further studied. |
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