Study On Improving Plant Height Of Huangjiang Xiannuo By Editing Rice SD1 Gene With CRISPR/Cas9 Technology

Rice plant height is one of the important plant types that affect yield.A traditional rice cultivar Huanjiang Xiangnuo in Guangxi hechi is known being a high quality glutinous rice.However,its high stalk increases its susceptibility to lodging which leads to grain losses and low production.Thus,shortening the plant height may enhance the plant architecture and increase the resistance to lodging,thereby improve the nutrient utilization efficiency and yield.The SD1gene,is the main gene controlling plant height that has been widely used to improve plant height owing to its insignificant negative genetic effect.The rise of Clustered Regularly Interspaced Short Palindromic Repeats,CRISPR-associated proteins9（CRISPR/Cas9）gene-editing technology provided new opportunity for the rapid and efficient breeding of new varieties with high-quality.In this study,we used CRISPR/Cas9 technology to knock out the SD1 gene to obtain the Huanjiang Xiangnuo mutant line with reduced plant height.The results obtained are as follows:1.Using Nipponbare SD1 gene as a reference sequence,the SD1 gene sequence of Huanjiang Xiangnuo parents was cloned and five high-efficiency target sites at exon 1 and exon 2 of SD1 gene was designed through the CRISPR-GE online tool.Two-target vector 1（CRISPR/Cas9-SD1-1,target sites1,2）and three-target vector 2（CRISPR/Cas9-SD1-2,target sites 3,4,5）were constructed.The constructed vector was verified by PCR,sequencing,and restriction digestion to ensure the accuracy of the recombinant vector.2.Agrobacterium-mediated transformation of Huanjiang Xiangnuo,32 and21 transformed plants by vector 1 and vector 2 were obtained respectively,and27 and 17 positive single plants were obtained by PCR screening,and the positive transformation rate reached 84%（27/32）,81%（17/21）.The target site sequencing analysis of all positive plants showed that the two target sites of vector 1 mostly showed the insertion and replacement of individual bases and a small number of base deletions.There was no mutation type of large fragment deletion,and target site 2 is more efficient than target site 1,obtained 8 multiple mutants with dual targets;and most of the positive plants transformed by vector2 are of base substitution type.There are 8 mutants at target 3,only one mutant at target 4,and at target 5 a total of 10 mutant strains,no three-target multiple mutants were obtained.3.We investigated the agronomic traits in T₀generation of transformed mutants by vector 1,The results showed that the average plant height of Huanjiang Xiangnuo mutant plants was 82.52 cm,while the average plant height of wild type was 140.88 cm.This suggested that the average plant height decreased by 58.36 cm（41%）with a very significant difference（P<0.01）.Agronomic traits such as spike length,effective spike number,flag leaf length and flag leaf width decreased compared to the wild-type parent;however,there was no significant difference in other agronomic traits（thousand-grain weight、Ratio of grain length to width）.4.The T₁generation transformed mutant lines of vector 1 were screened and identified for T-DNA-free plants.Five mutant lines were identified,a total of 180 plants were identified,and 42 mutants without transgenic components were successfully screened.Some T-DNA-free plants were sequenced and identified,and the sequence mutation results obtained were consistent with the T₀generation.5.The enzyme-linked immunoassay was used to determine the activity of GA₁,GA₃,GA₄and GA_20oxenzyme content of the T₁mutant strains,and the results showed that the contents of GA₁,GA₃,and GA₄of the tested mutants（2-5-6、2-10-6、2-11-12）all decreased,and the content of GA_20oxenzyme decreased significantly.The plant height of the tested mutants was measured,and the plant heights of the tested mutants were all semi-dwarfed,indicating that the plant height traits are affected by endogenous active GAs.q RT-PCR detected the relative expression of SD1 gene in wild-type and mutant lines,and the results showed that the expression of SD1 gene in mutant strains were all down-regulated to varying degrees,indicating that SD1 gene mutations affected its expression.6.The agronomic traits of some T-DNA-free lines in the T₁generation showed that the average plant height of the tested lines was significantly cut down and other agronomic traits were also affected.In conclusion,this study successfully constructed dual target vector 1 and triple target vector 2 of SD1 gene,created Huanjiang Xiangnuo sd1 mutant,and screened out semi-dwarf mutant lines without T-DNA.This research provides a certain reference for the cultivation of high-quality and high-yielding new rice varieties,and is of reference significance for further better use of CRISPR/Cas9technology to improve plant height.