| Food security has always been one of the major international issues of the world’s greatest concern.In recent years,the improvement of food crop production led by traditional breeding technology has gradually stalled.Combined with the problems of global food supply chain disruption caused by the Russia-Ukraine war and the COVID-19 pandemic,it is increasingly unlikely that the world will achieve the goal of "zero hunger" by 2030.Transgenic technology,as a modern emerging biotechnology,can stabilize overexpression of stress-resistance/high-yield related genes,which can effectively improve or even solve the problem of global food security.As a staple food in more than 100 countries,rice feeds more than half of the world’s population and plays a crucial role in solving the problem of food in poor regions such as Asia and Africa.Therefore,the exploration of key genes related to the improvement of physiological traits and the cultivation of new strains with excellent yield traits by combining transgenic technology with rice breeding is undoubtedly a breakthrough new direction to achieve the goal of "zero hunger".Ta NOX7,a member of RBOH family gene,was newly discovered in our laboratory earlier,and the expression level of this gene was the highest in young ears,which may be related to the development and fertility of wheat.Corresponding overexpressed lines and silenced lines were created in rice,and the data of development cycle and agronomic traits further verified that Ta NOX7 was closely related to plant development and function.On the basis of the above results,this study continued to analyze and evaluate the genetic stability and safety of NOX7-5 and NOX7-6 transgenic rice lines with Ta NOX7 gene by combining physiological and molecular tests.Main research results are as follows:1.Transgenic rice lines NOX7-5 and NOX7-6 have integration stability.T-DNA insertion sites of T1-generation transgenic lines NOX7-5 and NOX7-6 were separately amplified by TAILPCR technique,and the amplified flanking sequence fragments were compared with the base sequence of rice original genome,and the insertion copy number and flanking sequence at the insertion location were obtained successfully.Through specific PCR technology,genetic material from T1-T3 NOX7-5 and NOX7-6 lines were detected for the main exogenous gene Ta NOX7,GUS and Hyg.Obvious bands were found in the corresponding bp length interval of each gene primer.Therefore,exogenous genes Ta NOX7,GUS and Hyg have been successfully integrated into the rice genome and can be stably inherited in different generations.2.The target genes in transgenic rice lines NOX7-5 and NOX7-6 are stable in expression.According to q RT-PCR,the relative expression levels of exogenous genes Ta NOX7,GUS and Hyg in T1-T3 NOX7-5 and NOX7-6 lines were determined.The results showed that the above three genes were all highly expressed in multi-generation transgenic rice lines compared with WT,indicating that exogenous genes Ta NOX7,GUS and Hyg were stably expressed in transgenic rice lines.3.Transgenic rice lines NOX7-5 and NOX7-6 have the stability of improved physiological characters.The germination rate and emergence rate of WT and transgenic strains in multiple generations were calculated,and it was found that the germination rate and emergence rate of Ta NOX7 transgenic rice seeds were generally faster.The whole growth cycle of multigeneration transgenic rice lines from seed germination to maturity was tracked and recorded,and the comprehensive agronomic traits of rice at maturity stage were observed and analyzed.Further analysis has found that compared with WT,NOX7-5 and NOX7-6,which are overexpressed,have a shorter vegetative growth cycle and a longer reproductive growth cycle.Moreover,analysis on yield-related agronomic traits has also found that overexpressed strains are generally superior to WT.Therefore,it is preliminarily concluded that Ta NOX7 gene transfer can promote seed growth,shorten the total growth cycle,and improve the development of NOX7-5 and NOX7-6.Ultimately,it is estimated that NOX7-5 and NOX7-6 can increase the yield of a single plant by 21.83 % and16.38 %,indicating that Ta NOX7 overexpressed lines have stable physiological character improvement function.4.Transgenic rice lines NOX7-5 and NOX7-6 are environmentally safe.Through the determination and evaluation of pollen viability of WT and T2 transgenic rice,it is found that the transmission mode and transmission ability of Ta NOX7 transgenic rice are not significantly different from that of wild type under appropriate environment,indicating that genetically modified lines NOX7-5 and NOX7-6 are consistent with the survival competitiveness of receptor control wild type lines.Genetic material was extracted from 100 m weeds around the planting area of transgenic rice at flowering stage,and specific PCR was performed for the target genes Ta NOX7,GUS and Hyg.The results showed that there was no target band in the bp length interval corresponding to Ta NOX7,GUS and Hyg.The results indicated that the genetic material of transgenic rice was less likely to transfer to other plants especially gramineous plants in the surrounding environment.The insect population and weed population in the surrounding environment of transgenic rice and wild type were recorded by trapping method,and it was found that there was no significant difference.It was preliminarily judged that transgenic rice strains would not change the population and quantity of organisms in the field,which further explained its environmental safety.5.Transgenic rice lines NOX7-5 and NOX7-6 are safe for human health.The bioinformatics method was adopted,referring to the announcement of the Ministry of Agriculture,"Bioinformatics Analysis Method for Detection of Toxicity and Antitrophic Effects of Exogenous Proteins in the Food Safety of Genetically Modified Organisms and Their Products" and "Bioinformatics Analysis Method for Detection of Allergenic Effects of Exogenous Proteins in the Food Safety of Genetically Modified Organisms and their Products".The toxicity,antitrophic and sensitization of the T-DNA proteins Ta NOX7,Hyg and GUS were predicted in multiple corresponding databases.The results showed that there was no significant sequence similarity between the toxic,antitrophic and sensitizing sequences and the Ta NOX7,Hyg and GUS amino acid sequences.NOX7-5 and NOX7-6 transgenic rice lines with Ta NOX7 gene were determined to be safe for human health.In summary,the study demonstrated that exogenous genes Ta NOX7,GUS and Hyg have been stably integrated into the genome of rice and can stably express their physiological improvement ability.In terms of safety,the survival competitiveness of NOX7-5 and NOX7-6 transgenic rice has not changed,and the possibility of gene leakage to other plants is small,and will not change the population of plants and animals in the field.Meanwhile,bioinformatics detection has not found that the expression of exogenous genes has harmful effects on human body.Therefore,Ta NOX7 transgenic rice NOX7-5 and NOX7-6 are genetically stable and environmentally friendly,which has certain application value as an improved physiological trait crop. |
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