Mechanism And Application Of Fine-Tuning EUI1 To Improve The Performance Of Heading Of Rice Male Sterile Lines

Male sterile line（two-line male sterile line or three-line male sterile line）is often accompanied shortening of the uppermost internode,leading a part of the panicle is encased in the sheath of the flag leaf and blocks pollination.In production,a large amount of synthetic gibberellin has been used to improve the heading capacity of male sterile lines.However,the use of gibberellin not only reduces the quality of hybrid seeds,but also increase the cost.So,it is the key solution to improve the heading ability of sterile line from heredity.Breeders found,eui gene can significantly improve the elongation ability of the upmost internode,which is called"the fourth genetic element of hybrid rice".Previous study showed that eui1 had a better ability to elongation of the uppermost internode than eui2,So,eui1is widely used to improve the panicle exertion.However,eui1 can’t be used in production because of the extremely elongation of uppermost internode at the heading stage.Therefore,fine-tuning EUI1 to make the uppermost internode extend moderately is the key for eui1 germplasm to be applied in production practice.Here,we used a new plant adenine base editor.It can precisely edit the base pair of the target gene,Utilizing the plant adenine base editor tool,we created two transgenic mutants with different base substitution types,eui1-1 and eui1-2;Here we summarize the findings:1.We used the adenine base editing system to edit EUI1 and obtained homozygous mutants eui1-1and eui1-2 with two different base substitution types（C815S was the genetic background）,both of which changed the original folding pattern of EUI1 protein.2.Phenotypic analysis showed significant elongation of the mutants’uppermost internodes.Further analysis of paraffin sections of the mutants’uppermost internodes showed that elongation of the mutants’uppermost internodes was due to significant elongation of cell length.Through the detection of the headset rate of the mutant,we found that the headset rate was reduced significantly.3.Tissue expression analysis showed that EUI1 was a constitutive expression,and the highest expression level was found in young panicle.The transcription level and protein abundance of EUI1 in the mutants were significantly lower than that of wild type,and further detection of the enzyme activity of EUI1 in the mutants showed that the enzyme activity was also reduced significantly.4.EUI1 is a gibberellin inactivation enzyme,which inactivates active gibberellin produced in young panicles,thus inhibiting gibberellin flow from panicle to the upper internode,thus inhibiting the elongation of the upper internode.We detected the content of active gibberellin in young panicles of mutants,and found that the GA₁,GA₃ and GA₄ in mutants were increased significantly.5.In the seed production experiment,we found that the mutants eui1-1 and eui1-2 still maintained a low panicle wrapping rate,and the outcrossing rate was increased significantly.