Transcription Factor NST3 Gene Editing And Over-expression Vector Construction In Brassica Napus L.

Rapeseed(Brassica napus L.)is one of the most important oil crops in China and is widely cultivated in Europen,Austrilian and North American.In our country,the main causes of affect the rapeseed production is Sclerotinia disease and Lodging.In former and our research both indicated thatthe lignin content is related to the disease resistance and lodging resistance.The improvement of the cell wall intensity by the cell lignification can not only enhances the disease resistance of plants,but also improves the lodging resistance.The Sclerotinia disease and lodging are occurred easily in the later stage of rapeseed production.Furthermore,in our research we found that the higher content of S lignin monomer,the more resistantance to lodging Sclerotinia sclerotiorum.In our research,we conducted the expression analysis of the four members of NST3 gene in different organs of rapeseed,gene structure analysis online,bioinformatics analysis of encoded proteins,constructing CRISPR-Cas9 gene editing vector and over-expression vector of BnaA09g24190 D.CRISPR-Cas9 vector was successfully constructed and transformed into B.napus,and positive transgenic seedlings were obtained.DNA extracted from positive transgenic B.napus seedlings,PCR amplification and target gene sequencing.The main results of this study are as follows:1.As for the 4 menbers of NST3 family in B.napus,expression of BnaA09g24190 D and BnaC05g24890 D were higher than those of the other two members.The expression of BnaA09g24190 D was higher than BnaC05g24890 D in roots,stems,buds,flowers,pods and seeds.In roots,the expression of BnaA09g24190 D was almost 5 times than that of BnaC05g24890 D.The expression ofBnaA09g24190 D was almost 2.5 times higher than that of BnaC05g24890 D in stem.BnaA05g17950 D and BnaC05g28390 D were lowly expressedin rapeseed,and were not expressed in cotyledons,leaves and buds.Based on the gene expression analysis,we assumed that BnaA09g24190 D and BnaC05g24890 D play a major role in the regulation of secondary cell wall synthesis and regulation of lignin monomer composition,especially BnaA09g24190 D.2.The bioinformatic analysis indicate that the full length of BnaA05g17950 D is1819bp,the CDS is 927 bp,which has 4 exons and 3 introns.The full length of BnaA09g24190 D is 1776bp;the CDS is 1095 bp,which has 3 exons and 2 introns.The full length of BnaC05g24890 D is 1832bp;CDS is 1116 bp,which has 3 exons and 2introns.The full length of BnaC05g28390 D is 2146bp;CDS is 978 bp,which has 5exons and 4 introns.The protein analysis showed that protein encoded by the four members have 308?364?371?325 amino acids,respectively,molecular weight of which is between 35.75~42.99 KD.The acidic protein isoelectric point was less than 7.In secondary structure,alpha helix accounted for 24.45%~29.54%,?-turns accounted for 8.36%~8.77%.40.62%~48.35% for random coil,extension chain accounted for17.25%~21.23%.They have a typical conserved NAM domain,and their conserved domains can predict the tertiary structure.3.In the transgenic research,22 lines positive transgenic plants were obtained.In the transgenic plants,the editing efficiency of different menbers was different.In 22 lines positive plants,BnaA05g17950 D was edit in two plants,and the efficiency of editing was about 9.09%.BnaA09g24190 D was edited in 11 lines the efficiency of editing is 50%.BnaC05g24890 D was edited in 8 lines,the efficiency of editing is36.4%.BnaC05g28390 D was edited in 4 lines,and the efficiency was 18.2%.The four mebmers were edited at the same time is No.3,No.22 lines.BnaA09g24190 D and BnaC05g24890 D are edited in No.1 ? No.7 lines.In the No.9 lines of rapeseed transgenic plants,BnaA09g24190 D,BnaC05g24890D and BnaC05g28390 D have been edited.BnaC05g24890 D and BnaC05g28390 D of No.12 transgenic rapeseed were edited simultaneously.And that apply to BnaA09g24190 D and BnaC05g24890 D of No.17 transgenic rapeseed.The results obtained by CRISPR-Cas9 editing can be classified into three categories: the target site and its adjacent base deletion,insertion,deletion and insertion.4.As for the four members,BnaA09g24190 D plays the main role in the regulation of rapeseed secondary cell wall synthesis and lignin monomer composition,and weconstructed the over expression vector of BnaA09g24190 D and transformed it into rapeseed Wester.In the follow-up work of this experiment,the changes of lignin content in the transgenic seedlings of CRISPR-Cas9 editing and over expression,the identification of resistance to Sclerotinia sclerotiorum and lodging need to be followed by the laboratory.