| Corn is a high-yield food crop in the world.Its grains and stems are rich in protein,fat,vitamins,trace elements,cellulose,etc,which has broad development and application prospects.Fragrance is an important quality trait in food.It can not only improve consumer acceptance,but also improve the value attribute of the product.There are more than 100 volatile compounds were reported responsible for fragrant rice,2-Acetyl-1-pyrroline(2AP),a principal aroma compound has been detected in all aerial plant parts of scented rice.Studies have found that the production of aroma is related to betaine aldehyde dehydrogenase 2(BADH2).When the function of BADH2 is lost or expression is reduced,2AP will accumulate and produce aroma.In addition to rice,natural aromatic varieties have also been found in sorghum,soybeans,mung beans,cucumbers and coconuts,but currently aromatic germplasm has not been identified in maize.CRISPR/Cas gene editing technology can achieve rapid improvement of crop traits through precise editing of target genes,and has now become a new method of crop breeding.In this study,the one normal line,Zheng 58,and three waxy lines,N355,XCW175 and LN005 M,were used as experimental materials.Three CRISPR/Cas vectors were constructed to edit the aroma gene BADH2,which was transformed into maize through an Agrobacterium-mediated maize genetic transformation system.The immature embryos finally obtained fragrant corn in different inbred lines,creating fragrant corn germplasm resources.The specific research content and results are as follows:1.The protein sequence of Os BADH2 was obtained from NCBI website,and then perform a Blast comparison with the maize B73 protein database,and finally found that there are two Os BADH2 homologous genes in maize,they are Zm00001d050339 on chromosome 4 and Zm00001d032257 on chromosome 1.We named them ZmBADH2 a and ZmBADH2 b,respectively.Through q RT-PCR analysis,it was found that ZmBADH2 a and ZmBADH2 b were expressed in roots,stems,leaves and seeds of different pollination periods.2.Sequencing the first and second exons of Zheng 58,N355,XCW175 and LN005 M,and then comparing them with the maize B73 database to avoid SNPs affecting editing efficiency when designing targets.In order to verify the influence of ZmBADH2 a gene and ZmBADH2 b gene on fragrance substances,six sgRNAs were designed,namely sgRNA1,sgRAN2,sgRNA3,sgRNA4,sgRNA5 and sgRNA6.The p0593 vector targeting both ZmBADH2 a and ZmBADH2 b genes was constructed with sgRNA1 and sgRNA2 as targets,the p0195 vector targeting ZmBADH2 a was constructed with sgRNA3 and sgRNA4 as targets,and the p1801 vector targeting ZmBADH2 b was constructed with sgRNA5 and sgRNA6 as targets.4.The immature embryos of Zheng 58,N355,XCW175 and LN005 M were genetically transformed through the Agrobacterium-mediated maize genetic transformation system,and ZmBADH2 a edited plants,ZmBADH2 b edited plants and ZmBADH2 a and ZmBADH2 b were simultaneously edited in Zheng 58 and XCW175.The editing rate of Zheng 58 was 76.92%-100%,and the editing rate of XCW175 was 45.83-100%;the editing rate of ZmBADH2 a was obtained in N355 and LN005 M,the editing rate of N355 was 81.58%,and the editing rate of LN005 M was23.08%.5.Three different strains were selected from the E2 homozygous plants of zmbadh2 a mutant,zmbadh2 b mutant,and zmbadh2 a and zmbadh2 b double mutants,and use GC-MS method to detect the content of aroma substance 2AP in the seeds of different strains.The results showed that the presence of 2AP could not be detected in zmbadh2 a mutant,zmbadh2 b mutant and wild-type seeds;in the fresh food and mature seeds of zmbadh2 a and zmbadh2 b double mutants,different levels of2AP(0.028-0.723 mg/kg).6.The plant height,leaf number,and flowering time of the E2 mutant line were counted.Compared with the wild type,the plant height,leaf number,and flowering time of the mutant line were not significantly different.Therefore,there was no obvious influences on its main agronomic traits of the mutant line after the mutation of the BADH2 gene. |
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