Research On ALS Gene Editing Technique Of Maize Backbone Inbred Line Based On Pollen Tube Pathway

In this study,the gene editing of the acetylsalicylic acid synthase ALS gene in several important maize backbone inbred lines was studied by using the pollen tube introduction technique mediated by membrane peptide,which was used as the material of maize backbone inbred line,aiming at the preliminary establishment of maize gene editing technology system,which was not dependent on genotype,and lay a foundation for maize molecular breeding.Through the screening of large-scale seedling herbicides,the offspring of anti-tribenuron-methyl plant were obtained,and the target sequencing results preliminarily confirmed that Gene Sentinel editing could be achieved by introducing DNA into pollen tube,and the fixed point mutation of ALS gene could be used for herbicide resistance screening.Using the edited herbicide resistance gene as the screening marker,the identification efficiency of the gene editing offspring of pollen tube introduction method was greatly improved.In order to select suitable receptor materials and herbicide screening concentrations during the cultivation of herbicide resistant mutant materials,the sensitivity of various maize inbred lines to tribenuron-methyl and imazapic were studied using 7 maize inbred lines C92,Jing724,Jing2416,B73,Zheng58,HZ178and 178.We measured the inhibition rate of primary root length,seedling growth and acetolactate synthase?ALS?activity of each inbred line under different herbicide concentration.The results showed that the inhibition rate of primary root length and seedling growth of each inbred line increased obviously along with the increase of the concentration of two herbicides.The ALS activity of each inbred line under herbicide gradients decreased in varying degrees.The sensitivities of 7 inbred lines under two herbicides were:C92>Jing724>Jing2416>B73>Zheng58>HZ178>178.All the inbred lines were more sensitive to imazapic than tribenuron-methyl.To sum up,compared with B73,Zheng 58,178 and HZ178,the inbred lines of C92,Jing 724 and Jing 2416 are more sensitive to tribenuron-methyl and imazapic,and are suitable as receptor materials for cultivation of anti-herbicide mutant.The screening concentrations of tribenuron-methyl and imazapic were 334mg/L and 120mg/L respectively.The plasmid?pBUE911?harboring the editing elements of maize ALS gene was introduced into maize inbred line by pollen tube introduction method.22 T₀ resistant lines were obtained by screening with herbicide Tribenuron-methyl?334mg/L?,and single mutation of the ALS gene target was proved by molecular detection and sequencing verification,the sequencing analysis of the target point of the mutant self-intersecting offspring proves that the editing mutation can be effectively inherited.Seven mutant lines of T2 generation were choosed to identify the resistance of tribenuron-methyl and measure the activity of acetyl lactate synthase?ALS?.The results showed that,compared with the control,under the treatment of the tribenuron-methyl,the morphological indexes of root length,lateral roots,fresh/dry weight,plant height,stem thickness and leaf area of mutant lines in different growth periods were significantly increased,showing better growth status,and the activity of acetyl lactate synthase in mutants did not decrease significantly.The above results show that the change of amino acid obtained by the gene editing of ALS targetcan confer the maize inbreds some resistance to the tribenuron-methyl herbicide.