Construction And Genetic Transformation Of BnTT8 Gennome Editing Vectors In Rapeseed(Brassica Napus L.)

CRISPR/Cas9 gene editing is the latest genome editing technology developed in recent years,which enables knockout,replacement and site directed mutagenesis of a specific segment of genes.The TT8 gene is considered to be a key gene in the flavonoid synthesis pathway,which regulates the synthesis of a variety of seed coat pigment substances in the flavonoid synthesis pathway.Considering the close phylogenetic relationship between Arabidopsis and Brassica,TT8 homologous genes may have similar biological functions in Brassica napus L.Genome editing based on CRISPR/Cas9 system,as a genetic engineering technology developed in recent years,enables deletion,insertion or substitution of specific bases in a DNA sequence,resulting in loss or alteration of gene function,and provides a reliable technique and ways to deeply explore the biological function and molecular mechanism of specific gene.In order to establish a highly efficient genetic transformation system using Zhongshuang 11（ZS11）as the recipient material for convient genome editing in Brassica napus and to simultaneously obtain a number of excellent germplasm resources with yellow seed phenotype,two dual-target genome editing vectors targeting the BnTT8 genes in ZS11 genome were constructed using the CRISPR/Cas9 system in this study.Subsequently,Agrobacterium-mediated transformation of these vectors into ZS11 genome using petiole and hypocotyl as explants were carried out,and three factors influencing genetic transformation efficiency including preincubation time,infection time and hygromycin concentration based on our old procedures were explored.Finally,the regenerated resistant transgenic plantles were subjected to molecular detection and targets editing were analysed by sequencing.The main results of this study are as follows:1.BLASTn homology search against the reference genome sequence of Brassica napus（v4.1）revealed that there were two functional copies of TT8 homologous genes,Bna A09g22810D and Bna C09g24870D,named Bn A09.TT8 and Bn C09.TT8b in this study.The coding regions of these two sequences were both 1566 bp in length,encoding 521 amino acid residues with 97.89%amino acid sequence identity,and had similar structural composition and physicochemical properties.To enable simultaneous knockout of the two BnTT8 homologs,four sites were selected in the 5’end exons(1^stand 2^nd)and four sg RNA seed sequences were designed according to the principles,eventually two dual-target CRISPR/cas9 genome editing vectors,p CAMBIA1300-BnTT8-T12 and p CAMBIA1300-BnTT8-T34,were constructed using p CAMBIA1300-P35S:Cas9 as backbone.2.Agrobacterium-mediated transformation was carried out using ZS11 the recipient.It was found that the callus induction rate and shoot differentiation rate of cotyledonary petioles and hypocotyls could reach the maximum values after preincubated for 3 d,with an averaged callus induction rate 93.33%,45.00%and a shoot differentiation rate 53.57%,18.64%,respectively.With the infection liquid OD₆₀₀=0.6,the shoot differentiation rate of hypocotyl explants infected for 2 min was the highest 47.46%,while the cotyledon petioles needed 5 min infection and reached the highest 71.28%.Under these conditions,the explants had less browning and less Agrobacterium contamination.The results of screening pressure test showed that the positive rate could be effectively ensured by suplymenting 30 mg/L hygromycin in the medium at the stages of callus induction and shoot differentiation,so it was an optimal screening concentration for cotyledonary petioles and hypocotyls of ZS11.3.A total of ten resistant regenerating seedlings were obtained in this study,and the results of PCR with individual genomic DNA showed that all ten transgenic plantlets had the expected fragments of Hyg R and Cas9,six of which were derived from the hypocotyl explants and two from the cotyledon petioles.Furher analysis by sequencing demonstrated that seven transgenic positive seedlings had base substitutions within or near the T4 target in the T0 generation,all of which were single or multiple base substitution types,deletion or insertion was undetectable.Among them,plant#1and#7 produced substitutions at T4 targets in both BnTT8 homologs,while the remaining five plants produced substitutions only in one of them.In all cases of substitutions,four（#1（a2）,#3,#6,#9（a1））would result in premature termination of Bn A09.TT8 protein translation,three（#4,#7,#9（a2））and one（#7）would change the amino acid sequences of Bn A09.TT8 and Bn C09.TT8b,respectively.