Cloning Of MLO Gene In Hevea Brasiliensis And Construction Of Gene Modified Vectors

Hevea brasiliensis are important energy crops.Powdery Mildew is one of the most important leaf diseases in hevea brasiliensis production.Disease resistance breeding is one of the effective methods to control plant diseases.Gene modification technology(Clustered regularly interspaced short palindromic repeats-associated endonuclease,CRISPR/Cas)can be used to inactivate the susceptible genes of host plants or negatively regulated plant disease resistance genes,thus producing disease resistant varieties.Therefore,it is the premise that cloning the negative regulatory resistance gene of hevea brasiliensis against powdery mildew is the premise,and the construction of CRISPR/Cas9 technology application carrier is the key to cultivate disease resistant transgenic plants.Based on the research basis of the powdery mildew disease resistance negative regulation genes,the homologous gene Mlo was cloned from hevea brasiliensis to understand its expression during pathogen infection.After constructing the desired vector,this study attempted to use the CRISPR/Cas9 technology to target AtMlo2 transgenic plants of Arabidopsis dermatophyte powdery mildew mutant pad4.This gene is homologous to the HbMlo2 gene in rubber.It will pave the way for the later development of hevea brasiliensis resistant to powdery mildew through CRISPR/Cas9 technology.The main findings of this study are as follows:1.The full-length HbMlo2 gene was obtained by a piecewise cloning method.The gene sequence analysis showed that the DNA sequence of HbMlo2 gene was 3435bp in length,and the gene cDNA sequence was 1725bp in length,which encoded 574 amino acids.The homology of the HbMlo2 gene,same with the MLO family protein(AQX37913.1)of Hevea brasiliensis,reached 98%.Through bioinformatics analysis,the protein 14-516aa encoded by HbMlo2 gene had a typical Mlo superfamily conserved domain,eight transmembrane structures,and no signal peptide.2.Hevea brasiliensis brucella was used to inoculate the rubber leaves of Bronze Age and cultured to the grade index of 0,1,2,3,4 and 5 to extract RNA.The quantitative analysis of HbMlo2 gene expression in the pathogenic process of P.infestans infected leaves was analyzed by fluorescent quantitative PCR.The results showed that HbMlo2 gene was an up-regulated gene,and its relative expression at different times had a tendency of rising first and then decreasing,and it was a gene that negatively regulates disease resistance.3.After screening negatively regulated genes against powdery mildew,three negative resistance genes Mlo2,EDR1 and LFG were selected.Three sgRNA/crRNA sequences were designed and synthesized for Arabidopsis three homologous genes AtMlo2,AtEDRl and AtLFG.The restriction enzyme digestion successfully synthesized the vector pRD278 containing the three gene AtMlo2/EDR1/LFG sgRNA/crRNA fragment,and its interior was designed with HindⅢ,SmaⅠ and XbaⅠ single enzyme digestion sites.A CRISPR/Cas9 vector pRD216 containing HindⅢ,SmaⅠ,XbaⅠ,EcoRⅠ single enzyme digestion sites was successfully designed with pGEM-3Zf(+)as the basic skeleton.4.The pRD278 was linearized by restriction endonuclease and the AtMlo2 sgRNA/crRNA,AtMlo2/EDR1 sgRNA/crRNA,AtEDRl/LFG sgRNA/crRNA,AtMlo2/EDRl/LFG sgRNA/crRNA four-part sgRNA/crRNA fragment were successfully obtained according to the single restriction enzyme site.Four sgRNA/crRNA sequences were recombined into CRISPR/Cas9 vector pRD216 using enzyme recombination and ligation to obtain four vectors,ie,single gene AtMlo2 sgRNA/crRNA vector pRD286,double gene AtMlo2/EDR1 sgRNA/crRNA vector pRD287,double gene AtEDRl/LFG sgRNA/crRNA vector pRD288 and the three-gene AtMlo2/EDR1/LFG sgRNA/crRNA vector pRD280,laying the foundation for the development of CRISPR/Cas9 technology-modified negative resistance genes.5.pRD280,pRD286,pRD287 and pRD288 were subcloned into the corresponding restriction enzyme sites of plant binary expression vector pCAMBIA1300 respectively.Two restriction enzymes,PCR and other methods were used to verify that four CRISPR/Cas9 plants express recombinant vectors of pRD309,pRD310,pRD313 and pRD314 were successfully obtained.This study successfully constructed four Arabidopsis CRISPR/Cas9 genetic transformation vectors,laying the groundwork for the subsequent research on plant genetic transformation.6.We tried to use the single gene AtMlo2 modified vector pRD310 and the three gene AtMlo2/EDR1/LFG modified vector pRD309 as the plant transgenic vector.The vector was transferred to Agrobacterium tumefaciens EHA105,and the Arabidopsis inflorescence was invaded by Agrobacterium-mediated transformation.Through hygromycin selection and PCR validation,TO and T1 generation positive transformants containing the single gene AtMlo2 modified vector pRD310 were obtained,and the TO seed containing the three gene AtMlo2/EDR1/LFG modified vector pRD309 was obtained,but it did not germinate through the selection of hygromycina.