The Plant Height Improvement Of Xiangdaowan Based On CRISPR/Cas9 Gene Editing System

Plant height is one of the most important agronomic traits related to rice yield.The Xiangdaowan is a conventional variety of rice in Xinyang,which grows excessively tall and are easily flattened by wind and rain,resulting in low yield.Reducing plant height,to a certain extent,has an effect on the tolerance to high nitrogen fertilization and resistance to lodging.At the same time,it can also reduce the assimilation of rice stem;make the use of photosynthetic products more reasonably.The plant height of rice is affected by many factors,one of them is Gibberellin.SD1 gene coding of rice GA20 oxidase involved in regulating the generation of endogenous gibberellin in rice directly,and the sd1 mutation make rice become a semi dwarf state.Now,the sd1 gene has been widely used to reduce the rice plant height.In this study,we used CRISPR/Cas9 technique to construct the recombinant vector for oriented editing of SD1,which was transformed into the japonica variety Xiangdaowan.The sd1 mutants with reduced plant height were obtained.The results of this study are as follows:1.Two pairs of sgRNA were designed on exon 1 of SD1 gene using sgRNA online design software.Then,the CRISPR/Cas9-gRNA gene editing vector was constructed by linked pBWAH-Cas9i vector and gRNA.The recombinant vector was verified by PCR and sequencing.2.The callus of rice embryo was infected by Agrobacterium tumefaciens,and the regenerated transgenic plants containing T-DNA insertion were obtained.The mutation types were analyzed statistically by PCR and sequencing,and 7 lines of sd1 gene mutation rice were obtained.The main mutation type is the deletion of the 4th base in the upstream of the PAM region of target 2,where the deletion base is G;another few mutations to target 2 is a 5 bp lost at the position 3-7 before the PAM region.We also found a base insertion mutation,with a base A inserted in the position 4 at the upstream of the PAM region of target 2.All these mutations can cause the frameshift mutation of the target gene SD1,so that the SD1 gene coding region is terminated prematurely.As a result,the protein structure was damaged seriously,and the plant also exhitited a distinctly dwarf phenotype.3.56 lines of T₁ transgenic plants were detected by PCR.9 plants without T-DNA fragments were obtained,of which 3 were neither Cas9 nor HYG.PCR and sequencing analysis of the three plants without T-DNA insertion were performed,it was found that#13.7 is a heterozygous plant,and consistent with its parents,the editing site is a 5 bp lost at the position 3-7 before the PAM region of the target 2.The#13.3 is a heterozygous plant,too.But the editing sites are different from its parents.Presumably because its parents are chimera,the Cas9 protein can still play an editing role during their growth stage.#3.10 is a homozygous plant line,and its mutation site is identical to its parent plant,which has a 1 bp deletion at the 4th base in the upstream of the PAM region of target 2.The results in this study provide the experimental basis for the improvement of the Xiangdaowan,and also provide some research basis for the study of plant gene function using CRISPR/Cas9 gene editing technique.