| Flax is an important economic crop in China, the fiber is an important textile materials. In thetextile industry, lignin is the restriction factor of degumming process and quality of textile products, andthe removal of lignin process cause serious environmental pollution. Therefore, reduce of the content oflignin or change its composition by genetic engineering will solve the problem of pollution in paper andtextile industry from raw material. This work is one of the hot researches at home and abroad.In this study, full-length sequences of COMT and4CL gene were cloned and predicted bybioinformatics analysis, which are critical gene involved in flax lignin synthesis. In addition, researchwork on Agrobacterium mediated flax hypocotyl transformation by using RNA interference vector ofCOMT was carried through.The main results are as follows:(1) The full-length sequences of COMT and4CL were cloned using the RACE method. The full-lengthCOMT and4CL genes were registered in the GenBank, with GenBank accession number of KC832864and KC832865. The full-length sequence of COMT is1726bp, containing a complete open readingframe of1104bp. Molecular mass of the protein encoded by COMT is40kD; pI of the protein is5.7;leucine is vast majority of the amino acids composing component; α-helix is the mainly secondarystructure of the protein. The full-length cDNA sequence of4CL is1957bp, containing a complete openreading frame of1650bp. Molecular mass of the protein encoded by4CL is60kD; pI of the protein is5.3; leucine is vast majority of the amino acids composing component; random coil is the mainlysecondary structure of the protein; The result of Blastn with GenBank showed that COMT and4CL geneof flax have high homology with other species. Tertiary structure prediction of COMT and4CL areconsistent with the secondary structure prediction.(2) The research work on genetic transformation of flax lignin biosynthesis geneAgrobacterium mediated flax genetic transformation system was developed by COMT RNAinterference vector. The results are as follows: MS1medium was used as the genetic transformationmedium; pre-culture of hypocotyls persist for3d; hypocotyls after pre-culture were infected for30minin agrobacterium solution with OD value of0.5~0.8; co-culture continue for4d in MS1with AS at20℃; selective marker was added in6d after culture in MS1with ampicillin; ampicillin was used withconcentration of300mg/L; hygromycin was used with concentration of25mg/L when callus formationand differentiation and9mg/L when the root formation; hypocotyl browning rate was significantlyreduced in selective medium with5mg/L AgNO3.(3) The detection of regenerated seedlingsThe regenerated plantlets were detected by GUS assay and PCR. Results have showed that the purposegene had been integrated into the genome of flax. |
