| "Targeting induced local lesions in genomes TILLING",also known as "TILLING technology",is a brand new direction in the research of reverse genetics and directed mutation.The special feature of the method is that the chemical mutagen "Ethylmethane sulfonate(EMS)" is used to treat plant seeds and callus to produce a series of point mutations,and the CEL-Ⅰ is digested efficiently It is closely combined with the high frequency of chemical mutagenesis,which can quickly and effectively identify mutants from the mutant population,and provide novel germplasm resources for crop variety improvement.In this study,wine sorghum red tassel was selected as the test material,0.5% EMS was used to induce the treatment of sorghum seeds for 8 hours,and then planted in the incubator after germination.Effectively combining the observation of phenotypic mutation observations in field traits,constructing a mutant library,it was found that the sorghum plant height,leaf color and leaf shape,panicle type,yield factors,fertility and premature senescence mutations varied in varying degrees,and the mutation traits were abundant It laid the foundation for screening beneficial mutants;meanwhile,in the M1 and M2 generation lines,TILLING analysis was performed on the gibberellin receptor gene GID1 affecting sorghum,in order to expect to obtain drought-resistant mutants,and to create a new type of sorghum in drought resistance breeding Germplasm resources.1.Construction of mutant library and phenotypic analysisThe morphological variation of M1 mutants of sorghum plants was investigated in the field.Compared with the control,the seed setting rate,planting rate and emergence rate of theM1 generation treated with EMS were significantly reduced;in addition,the M1 and M2 variants were rich in morphological categories,mainly with 6 typical phenotypic variations.After investigation,it was found that some of the mutations in the M1 generation were physiological damage and returned to normal in the M2 generation.The total frequency of phenotypic mutations in the M1 generation was about 6.3%,Among them,there were 81 tillering mutants,with the highest mutation frequency,1.87%;leaf mutants,followed by 76 and 1.76%,respectively;54 plant height mutants,with a mutation frequency of 1.25%,and the highest plant height 135 cm,down to 45 cm;The fruiting situation of the M1 generation is also significantly different from the control,and beneficial mutations such as plant dwarfing have appeared.The morphological variation of M2 mainly manifested in trait mutations such as tiller number,plant height,fertility and premature senescence.Compared with the M1 generation,it has fewer mutation types,but each type of mutant plants increases and its mutation frequency is higher.There are 59 mutant plants with a mutation frequency of 5.36%.2.Analysis of drought-resistant physiological indexesAfter the EMS induction treatment,the average plant height of Red Gardenia sorghum was 73.55,the highest was 98 cm,the lowest was only 59 cm,and the coefficients of variation were 0.06 and 0.13,respectively.The average plant height is significantly lower than that of the wild type,and a certain dwarfing phenomenon has appeared.Because the plant height of drought-resistant plants is generally not high,plant dwarfing is one of the important agronomic traits for identifying the drought resistance of crops,and dwarfing of sorghum is one of the important indicators for breeding new drought-resistant varieties.The direct identification method in the field found that the plants M2-794,M2-333 and M2-746 had strong drought resistance in the field and reached the second level.The drought resistance of sorghum plants was identified by analyzing the physiological indexes of the red tassel sorghum M2 generation and the direct identification method in the field.the proline(Pro)content in M2-521,M2-619,M2-665,M2-727 and M2-794 was higher,the highest was M2-521,95.8 μg/g;the malondialdehyde content in M2-746,M2-33,M2-794,M2-574 and M2-333 was higher,the highest was M2-746,63.7nmol/g;the soluble sugar content in M2-794 and M2-333 was higher,87.2mg/g and 88.3mg/g respectively.Amongthem,the proline content of m2-333 is relatively low,while the content of other indicators is relatively average,but the drought resistance is relatively average.Statistical analysis found that the proline content,malondialdehyde,and soluble ugar content of M2-746 mutant plants were significantly higher than those of the control,which were 47.7 μg / g,63.7 mg/g,and49.1 mg / g,respectively.The plant height was significantly lower than the control,there was a certain dwarfing phenomenon,and there was no significant difference in leaf number and leaf size.Therefore,according to the content of proline,malondialdehyde,soluble sugar and so on,it was preliminarily judged that M2-746 plants had strong drought resistance,and the field drought resistance reached Grade II.3.Mutant bioinformatics analysisBioinformatics analysis results show that sorghum GID1 and millet(Setaria italica var)GID1 differ by only 1 amino acid in the A subdomain,4 amino acids in the B subdomain,and2 amino acids in the D subdomain are different.Sorghum GID1 also has similar conserved domains with other plants,and the differences are only individual or a few amino acid differences.Phylogenetic tree analysis revealed that the sorghum gibberellin receptor gene GID1 was closely related to that of pupae(Panicum hallii var.Hallii),corn(Zea mays),and millet(Setaria italica var).The prediction of the secondary and tertiary structure of the protein indicated that the α-helix in the amino acid sequence of the protein encoded by the GID1 gene was 30.6%,the extension chain was 14.1%,and the random coil sequence was 55.3%.It can be inferred that α-helix and random coil are a large number of structural elements of GID1 protein,and extended chains and β-turns are scattered throughout the protein.4.Detection of mutation sitesThe DNA pool was constructed by extracting the genomic DNA from the M1 and M2 generations of the mutagenized plants.According to the sequence of sorghum GID1 gene,five pairs of specific primers were designed,and the directed PCR amplification,CEL-Ⅰ enzyme digestion and capillary electrophoresis analysis were carried out on Sorghum GID1.One mutant plant M2-746 was screened out.After sequencing verification,eight base mutation sites were found,two of which were in the intron region,which would not affect the gene expression.There were one point mutation at 180 BP and 540 bp,two at 720 bp and 780 bprespectively,and three at 120 bp,with a mutation density of 1 / 9.44 kb.The 558 bp nucleotide of exon 1 of Sorghum GID1 gene was changed from TCC to TC-,among them,the C base was deleted,which caused a deletion mutation at the 186 th encoded serine,resulting in the deletion of serine.The related effects need to be further verified. |
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