Genetic Transformation Of Fertility Related Genes In Tobacco And Creation Of Sterile Materials

Plant male sterility(MS)refers to the abnormal development of male organs,but the normal development of female organs in the process of plant growth.It is an important character in the study of crop reproductive biology,closely related to the utilization of crop heterosis,and plays an important role in crop breeding and molecular biology.When tobacco male sterile line is directly applied in tobacco production,it is easier to achieve the production location of varieties.However,at present,there is only a single source of cytoplasmic male sterile lines which can be used in production,and only the cytoplasmic male sterile genes from N.suaveolens are successfully used,which leads to a great risk in the promotion and application of tobacco hybrids.In order to broaden the source of sterile genes in tobacco,the MS1 gene with fertility control function and TA29 gene as promoter were used in Arabidopsis thaliana and Capsicum annuum L,Through bioinformatics analysis and prediction of ntmss1 gene to control tobacco fertility,CRISPR / cas9 vectors of Ntms1 and TA29 genes were constructed by CRISPR / cas9 gene editing technology.Tobacco varieties K326 and TN90 were used for genetic transformation,identification and screening The mutants were selected and transformed.The mutation effect of the mutants was analyzed.It was confirmed that the two genes Ntms1 and TA29 had the function of controlling tobacco fertility.The new materials of tobacco male sterile line were obtained.The results are as follows:1.Bioinformatics analysis of MS1 geneBioinformatics prediction method was used to analyze the physical and chemical properties,secondary structure,hydrophobicity and phosphorylation sites of the proteins encoded by the two target genes.The results showed that the sequence similarity of the three proteins was 94.43%,the functional domains of the gene encoding products were all Ph D functional domains,and the secondary and tertiary structures of the proteins were very similar,containing phosphorylation recognition The results of phylogenetic tree analysis showed that the two genes had a certain genetic relationship.Therefore,it is speculated that ntmss1 gene and cams1 gene may have the same function.2.Cloning and vector construction of Ntms1 and TA29 genesThe specific primers were designed in primer 6.0 software by using the ntmss1 sequence downloaded from NCBI.The target gene CDs was amplified by PCR using the cDNA of tobacco varieties K326 and TN90 as template sequence.After electrophoresis,the product was recovered and connected with T vector.The sequencing work was carried out in Shaanxi boruide Biotechnology Co.,Ltd.and named Ntms1-1,Ntms1-2;TA29 base respectively Because(Gen Bank login No.LOC107824681)is downloaded directly on NCBI website,the clone is the same as MS1.Through the sequence analysis of three genes,the target sequence located in exon region and with high score was selected by target design website.The recombinant vector used Arabidopsis U6 promoter which can be efficiently used in dicotyledons,and the enhanced Ca MV which can efficiently express cas9 protein and hygromycin resistant gene was used 35 promoter,the two target sequences were connected with CRISPR / cas9 vector system,and finally four target gene knock-out vectors(MSG1(single knock Ntms1-2 gene),msg2(single knock Ntms1-1 gene),msg3(double knock Ntms1-1,Ntms1-2 gene),msg4(single knock TA29 gene)were constructed.3.Genetic transformation of recombinant vectorsIn the experiment,Agrobacterium tumefaciens mediated transformation of tobacco leaves was used,and K326 and TN90 were transformed into two experimental materials.The young leaves were disinfected,infected and cultured until resistant seedlings were grown.Finally,440 transformed plants were obtained,including 104 MSG1 vectors(the mutant was numbered with letter A),41 MSG2 vectors(the mutant was numbered with letter B),176 MSG3 vectors Strains(the mutant is numbered with the letter C),there were 119 MSG4 vectors(the mutants were numbered with the letter D).4.Screening and identification of transformantsPCR was used to design primers with cas9 gene and hygromycin respectively,and the positive rate of transformed plants was more than 80%.Primers were designed on both sides of the target site.The DNA sequence extracted from the transformed single plant was used as template to amplify the target site sequence.The PCR products were sent to the company for sequencing.The sequencing results were compared with the wild-type sequence analysis.The plants with changed bases were identified as mutant plants.The results showed that there were 95 MSG1 vectors(one of them was TN90),37 MSG2 vectors,166 MSG3 vectors and 109 MSG4 vectors.5.Analysis of mutation effectThe results showed that the gene expression of 18 mutant plants decreased significantly,which was 0.6 times higher than that of the wild type;the stamens of 2 mutant plants were lower than that of the pistil,the seed setting rate was about 50% of the wild type and the seeds were small and light;the proportion of active pollen of 11 mutant plants decreased significantly The content of MDA in leaves of 9 plants increased significantly,which was 1.42 times of that of the wild type.Through the analysis of genetic transformation and mutation effect,16 and 2 Ntms1 and TA29 transformation mutants were obtained respectively.The results showed that Ntms1 and TA29 had the function of controlling tobacco fertility.In this study,CRISPR / cas9 gene editing technology was used to confirm that Ntms1 and TA29 have the function of controlling tobacco fertility,and low fertility mutants were obtained.In the future,it is expected to develop a new sterile line of tobacco,which is of great scientific value and practical significance to promote the research of tobacco sterility and its utilization in tobacco production.