Molecular Cloning、Establishment Of CRISPR/Cas9 Technology System And Functional Expression Study Of Heteromeric ACCase Subunit Genes From Flax

Flax(Linum usitatissimum L.) is an important oil and fiber crop of flax.The content of human essential fatty acid α-linolenic acid in flax oil is over 50%.Acetyl-Co A carboxylase is a key enzyme that catalyzes the de novo synthesis of fatty acids in all organisms and is the rate-limiting enzyme in the process of fatty acid synthesis.Therefore,the research on ACCase gene of flax is of great significance to clarify the law of flax oil synthesis.In this study,Longya No.10,Zhangya No.2 and R2-17 three flax varieties with different oil contents were used as test materials to analyze the expression differences of flax ACCase gene in different tissues of these three varieties at different developmental stages and also the correlation between the expression level and the oil content in flax seeds;the full-length c DNA sequence of 4subunit genes of Longya No.10 heterogeneous ACCase was cloned,and 4heterogeneous ACCase of Longya No.10 were cloned by the CRISPR/Cas9 system The subunit genes were subjected to fixed-point editing,vector construction and genetic transformation,which initially verified the gene function.The main research results are as follows:1.Cloning and sequence analysis of flax ACCase subunit gene c DNA.Using Longya No.10 as the material,the c DNA sequences of the four subunit genes of flax ACCase were cloned,and their amino acid sequences were analyzed using bioinformatics methods.The results showed that the size of the c DNA fragments of the four genes of Longya No.10,acc A,acc B,acc C and acc D were 2364,1173,1584 and 1137 bp,respectively.And the fat coefficients of the proteins encoded by the four genes are relatively high,and no signal peptide is not a secreted protein.The three genes of acc A,acc C,and acc D each have a transmembrane domain,but the acc B gene has no transmembrane domain.The protein encoded by the acc A gene exists in the mitochondria and the nucleus,the acc B and acc D genes encode proteins in the chloroplast,but the acc C gene encodes proteins in the mitochondria.The tertiary structure of the protein encoded by the four genes of Longya No.10,acc A,acc B,acc C and acc D,is mainly composed of α-helix,β-turn and random coil and extension chain.It can be seen that the functions of the proteins encoded by the four genes of Longya 10,acc A,acc B,acc C and acc D,are similar to other oil species.2.The relationship between the expression pattern of flax ACCase gene and oil content.Taking the three flax varieties of Zhangya No.2,Longya No.10 and R2-17 as materials,the differences in expression of heterogeneous ACCase gene and homogenous ACCase gene in flax seeds of the three varieties were analyzed.The expression difference of flax ACCase gene in different tissues of three varieties of flax at different developmental stages,and the correlation between the expression level of flax seeds and oil content.The results showed that the expression levels of the four genes acc A,acc B,acc C,and acc D were significantly higher than those of the homogenous ACCase gene.And in the process of seed development and maturity,the expression levels of the four genes acc A,acc B,acc C,and acc D in the early stage of seed development were higher than those in the later stage of seed development,while the expression levels of the homogeneous ACCase gene reached the peak in the middle and late stages of seed development.The four genes,acc A,acc B,acc C and acc D,are expressed in different tissues at all periods in the three varieties R2-17,Longya No.10 and Zhangya No.2 and the expression levels in seeds are significantly higher Other organizations.The oil content of Zhangya No.2,Longya No.10 and R2-17 seeds in the early development stage(10,15,20,25,30 d)and the four genes acc A,acc B,acc C and acc D in the early development stage(5,10,15,20,25 d)There is a very significant difference in the relative expression of seeds,and there is a significant positive correlation.3.CRISPR/Cas9 system for site-specific editing,vector construction and genetic transformation of flax heterogeneous ACCase subunit genes.Successfully established the flax CRISPR/Cas9 technology system,and constructed 4 CRISPR/Cas9 recombinant knockout vectors CPB-CRISPR/Cas9-acc A,CPB-CRISPR/Cas9-acc B,CPB-CRISPR/Cas9-acc C and CPB-CRISPR/Cas9-acc D was transformed into Agrobacterium GV3101 by freeze-thaw method,and the strains with stable expression were selected.Longya No.10 was transformed by dipping method.A total of 64 accumulating genes were transformed into T0 generation fruits and 394 seeds,and 21 positive seedlings were obtained.acc B genes were transformed into a total of65 T0 generation fruits and 318 seeds and positive seedlings were obtained.Two strains,the acc C gene obtained 91 T0 fruits,599 seeds,11 positive seedlings,a total of 61 acc D genes,367 seeds,3 positive seedlings.